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Molecular Medicine – HAEM2000

Inaugural lecture – The revolution in medicine
How to produce a vaccine:
1) Sequence the viral RNA.
2) Identify important parts of the virus for the immune system response.
3) Isolate those genes.
4) Use them as either mRNA which our cells will use to make viral proteins which we can then develop
an immune response to or put them into a new harmless virus to act as a vaccine.

Chronic Myeloid Leukaemia

- Cancer characterised by puss-filled blood.
- Caused by a translocation between chromosomes 9 and 22 written as t(9;22).
- The ABL gene breaks from chromosome 9 and the BCR gene breaks from chromosome 22 and they
fuse.
- Chromosome 22, now containing BCR and ABL is known as the Philadelphia (Ph) chromosome.

- ABL is a tyrosine kinase – an enzyme involved in cell signalling.

- BCR-ABL is a functional but dysregulated tyrosine kinase and causes CML, and thus inhibiting this
tyrosine kinase (BCR-ABL) is the treatment for CML.
- Imatinib is a drug that inhibits BCR-ABL.
- Normally patients would die within 5 years of being diagnosed, but taking imatinib allows the CML
to be treated as a chronic disease. CML was changed from a fatal disease to a chronic one.

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The Organisation of the Nucleus – Week 1 Lecture 1
DNA
- There are more than 10 million species on Earth, of which most are single celled.
- In both single and multicellular organisms, the single cell contains all the hereditary information
(the entire genome can be found in a single cell) of that species.
- This hereditary information is found in a linear chemical called DNA (Deoxyribonucleic acid).
- DNA performs the same function regardless of species.
The genome comprises of all the genetic material that an organism possesses. It includes the DNA
sequence of each chromosome, as well as any DNA in organelles.
- Remember that the entire genome can be found in just a single cell of the organism, except for
mature red blood cells as these do not have a nucleus.
- The genome provides instructions to the body on how to live (maintain normal bodily processes -
homeostasis) through RNA and protein synthesis and for reproduction.
The transcriptome is the complete set of RNAs present in a cell, tissue, or organism. Its complexity is
mostly due to mRNAs, but it also includes noncoding RNAs. Note that not all DNA is transcribed.
The proteome is the complete set of proteins expressed by the entire genome. Some genes code for
multiple proteins, and thus the size of the proteome is greater than the number of genes.
The Central Dogma of Gene Expression
- Through the production of mRNA (transcription) and the synthesis of proteins (translation), the
information contained in DNA is expressed.

Two major types of cells:

- Prokaryotes – Have no distinct nucleus or specialized organelles.
Evolution then occurred and we get:
- Eukaryotes – Have membrane-bound organelles – most notably a nucleus.
The origin of the eukaryotic cell is the single most profound change in cellular organization during the
evolution of life on Earth. We do not know how this happened, but the most probable reasoning is the
Invagination theory. The cell membrane folded in on itself (invaginate) into the cytoplasm and established
itself around the nuclear material of the cell, forming a nucleus and later an endoplasmic reticulum. A
nucleus allows for greater specialization and diversity.

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Genome Arrangement in a cell
Bacterial genome arrangement:
- Bacterial DNA is a circular molecule that is a few million nucleotides in length.
- To fit in the bacterial cell, it must be compacted 1000 times (1000-fold):
Firstly through formation of loop domains – compacts 10 times.
Secondly through DNA super coiling within loops.

Eukaryotic genome arrangement:

- To gain complexity, eukaryotic organisms gained more and more DNA, and circular DNA become
linear to accommodate for more DNA.
- Eukaryotic species can contain one set of DNA (haploid) but usually contain two (haploid). Can
contain more.
- All chromosomes are linear and are located in the nucleus and must thus be highly compacted to fit
there.
- Each diploid cell contains about 2 meters of DNA.
- It is a problem to fit that much of DNA into a small space, and thus must be coiled 10 000 times.
Histone Proteins – 8 histone proteins (two of each H2A, H2B, H3 and H4) come together and form a
scaffold (backbone) around which DNA will coil. Histones are small, positively charged proteins.
- Since DNA is negatively charged (due to phosphate groups in its phosphate-sugar backbone),
histones bind with DNA very tightly. DNA wraps twice around the 8 histones.
- Nucleosomes- The basic unit of DNA packaging in eukaryotes. The unit of histone proteins plus its
DNA wrapped around it is a nucleosome.

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- A histone is a protein, which is a length of amino acids. The bulk of these amino acids from the
spherical protein, the amino terminal consisting of amino acids “waving around” are not part of the
spherical core and can be called histone tails. The N-terminal tails of core histones protrude from
the nucleosomes and can be modified enzymatically by biochemicals through:
- Acetylation
- Phosphorylation
- Methylation
- The histone proteins are in intimate contact with the DNA and thus modifications on the histone
tails will have profound effects on the DNA.
- Adjacent nucleosomes are joined by linker DNA which can be 8-114 base pairs (bps) long. This
makes it look like “beads on a string”. Nucleosomes are dynamic and thus can move its position
along the DNA strand – it does not have a fixed position. The length of linker DNA between
nucleosomes can change as the nucleosome moves. A given length of DNA anywhere in your
genome may contain more or less nucleosomes at different points in time. The chromatin structure
might be different due to the movement of the nucleosomes.

Packaging DNA into nucleosomes shortens it 7 times. 1 meter

of DNA will become a 14 cm “string of beads”. Much more
compaction is required and occurs as shown:

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Chromatin
- Eukaryotic DNA does not exist as naked DNA. It is always associated with histone proteins and other
non-histone proteins.
- Chromatin refers to DNA and its associated proteins (histone proteins and non-histone proteins).
- Prokaryotes do not have chromatin as they do not have histones.
- There are two types of chromatin:
1) Heterochromatin – Stains darkly as it is highly condensed. It is dispersed throughout nucleus but
concentrated at nuclear envelope and only found in Eukaryotes. Nucleosomes are closer to each
other and the length of linker DNA between nucleosomes is much shorter and the DNA is tightly
compacted.
2) Euchromatin – Stains lightly as it is less condensed. Nucleosomes are further apart from each
other, and the DNA is said to be in a relaxed conformation.
- Dark regions are called heterochromatic regions and light regions are called euchromatic regions.

- Cells can control access to their DNA by modifying the structure of their chromatin. Highly
compacted chromatin (heterochromatin) is not accessible to enzymes and transcription factors
involved in DNA transcription. Genes occurring in these heterochromatic regions will be silenced.
- The opposite is true for euchromatic regions. Genes in euchromatic regions can be accessed by
transcription factors and will thus be transcribed.

Chromosomes in the 3-D nucleus

- Chromosome arrangement in the nucleus is not random.
- Interphase chromosomes occupy a distinct part of the nuclear space known as chromosome
territories.
- Inter and intra-chromosome interactions are specific and affect nuclear functioning, including gene
expression.
- DNA-associated proteins (notably, histones) maintain order in the nucleus of eukaryotes.

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The Anatomy of a gene – Week 1 Lecture 2
Genes:
- Made up of DNA.
- Basic physical unit of inheritance.
- Contain information needed to specify traits.
- Humans have approximately 22 000 genes.
DNA consists of two chains of polynucleotide chains in an antiparallel
configuration (one strand is in the 5’ to 3’ direction and the other strand is
in the 3’ to 5’ direction).
- 5’ = upstream (beginning of gene)
- 3’ = downstream (end of gene)
Components of a gene:
- Exons – Portion of a gene that codes for amino acids (protein).
Usually short (100-200 bp in size).
- Introns – Non-coding regions of the gene between introns.
Larger than exons (About 3000 bp long but length varies).
Intron positions are usually conserved.
- We call our genes “Interrupted genes” because of introns and exons.

- Regulatory sequences/ regions are found upstream of the gene and include enhancers and
promoters. They regulate transcription of a gene to mRNA.

Enhancers – Activate the use of a promoter and by doing so control the efficiency and rate of
transcription for that gene. They are usually found upstream but can be found downstream or
anywhere along the gene. They must be present in the same strand of DNA as the gene being
transcribed. They can also inhibit transcription by acting as Silencers. Due to their ability to either
allow a gene to be transcribed or to block transcription, they are referred to the control region of a
gene.

Promoters – Special sequence that signals the start of the gene (TATA box). They are located
upstream at the 5’ end and they bind transcription factors. They facilitate recruitment and binding
of RNA Polymerase II and facilitate the start of transcription. It is a region recognised by the
enhancer.

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- When an enhancer is required before transcription it will loop over to attach itself to the
promoter/transcription factor region of the region forming a loop. The promoter will then recruit
transcription factors and RNA Polymerase II to start transcription.

Transcription occurs in 3 stages:

- Initiation – The signalling given by the promoter region to say that the transcription factors and
RNA Polymerase should bind.
- Elongation – RNA Polymerase begins to add RNA nucleotides in the sequence the DNA specifies,
therefore synthesizing the RNA molecule in the 5’ to 3’ direction. Transcription begins at the
beginning of the first exon.

- Termination – Terminator sequence signals for end of transcription. It is reached when the end of
the last exon is transcribed. The RNA sequence formed is called pre-mRNA.

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RNA Processing
- The pre-mRNA formed is now processed and introns are removed (spliced out).
- Splicing is a post-translational modification that results in the removal of introns/intronic
sequences. Mature mRNA is formed, and it is ready for translation.
- Conserved regions (regions that are usually always the same) of intron sequences are found
flanking the exon/intron boundaries and are called splice sites or splice junctions. 5’ splice sites are
found at the beginning of each intron and 3’ splice sites are found at the end of each intron.
- 5’ splice sites begin with GT (eg GTA or GTG) and 3’ splice sites end with AG (eg CAG). Recognition
of these bases allow for the removal of introns. We say they are conserved because they remain
the same and are thus easily recognisable. If there are changes to these sites, the intron may not be
spliced out and this may have consequences in the process of translation and further on in the
functioning of the protein that is supposed to be produced.

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Translation
- This is the decoding of mRNA and using its information to build a polypeptide (string of amino
acids).
- mRNA is made up of codons (3 nucleotides).
- Specific start and end codons mark the beginning and end of the amino acid sequence.
- Start codons mark the initiation of translation (such as AUG/ATG).
- Stop codons mark the termination of translation (such as TAA, TAG, TGA or UAA, UAG, UGA). Stop
codons have no corresponding tRNA’s.
- The start codon is not usually located on the first base of the first exon and the stop codon is not
usually located on the last base of the last codon. Thus, we have regions that have been transcribed
but will not be translated.
- The region at the beginning of the first exon (before the start codon) that is transcribed but will not
be translated is called the 5’ UTR (untranslated region).
- The region at the end of the last exon (after the stop codon) that has been transcribed but will not
be translated is called the 3’ UTR (untranslated region).

Once we put everything together, this is what is formed:

KNOW HOW TO DRAW THIS!!!

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- Promoter – TATA box.

- Start transcription at beginning of orange and end at end of orange.
- Start translation at start codon ATG and end at stop codon TAA.
- Exons in bold.
- Introns start with GT and end with AG.
- 5’ UTR and 3’ UTR are regions translated but not transcribed.

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DNA Structure and Replication – Week 1 Lecture 3
DNA replication is a highly regulated process to copy the genome (S phase of cell cycle). DNA exists as a
double-stranded polymer [made up of monomers called Nucleotides (and also RNA)].
- Nucleotides are made of 3 parts:
1) 5-carbon sugar (pentose). This means it is a sugar made up of 5 carbons.
It is ribose in RNA and deoxyribose in DNA.
2) Nucleobase attached to the 1’ carbon – guanine, adenine,
thymine (uracil in RNA) and cytosine.
3) Phosphate group attached to the 5’ carbon, which has a negative charge.

- DNA joins through a condensation reaction involving the 5’ Phosphate

group of one nucleotide and 3’ Hydroxyl group of another.

- When nitrogenous bases are attached to the 1’ carbon, they are called
Nucleosides. A Nucleoside combined with one or more phosphates is called
a Nucleotide. Nucleotides could also termed depending on the number of
Phosphate groups they are attached to, such as Adenosine monophosphate
(AMP), Adenosine diphosphate (ADP), Adenosine triphosphate (ATP) and so on.

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DNA Replication:
- The mechanism of DNA replication is
very similar across all organisms.

- 1) DNA unwinds and forms a replication bubble,

which has a y-shaped replication fork
(the junction of the unwound
molecules) on each side of the bubble. The DNA is now
separated into two single strands.

2) Each strand of the double-helix serves as a template

from which a new strand will be made, by pairing
complementary bases (G-C and A-T) with the old strand. Each new strand now contains one old
strand and one new strand, and this is known as semi-conservative replication.
Semi-discontinuous and bi-directional replication:
- Recall that DNA is anti-parallel – the two strands are parallel to each other but in the opposite
direction. One strand is in the 5’ to 3’ direction and the other is in the 3’ to 5’ direction.
- The enzyme responsible for DNA replication is DNA Polymerase (DNA is a polymer and Polymerase
builds the polymer).
- DNA Polymerase can only read DNA in the 3’ to 5’
direction, and thus builds the new strand in the
5’ to 3’ direction (because the new DNA strand must
be anti-parallel). Since the top strand is in the correct
direction (3’ to 5’), the DNA Polymerase can build
continuously, and this is called Continuous Replication.
- The problem now arises at the bottom strand, which is in the 5’ to 3’ direction. DNA Polymerase
must read it in reverse, but can only do so in short fragments, and these are known as Okazaki
fragments, and this is called Discontinuous Replication.
- Hence, in both the top strand (leading strand) and the bottom strand (lagging strand), DNA is still
being read in the 3’ to 5’ direction and synthesized in the 5’ to 3’ direction, but because they are
being synthesized in opposite directions, we say it is bi-directional.
DNA Polymerisation
- The joining of two nucleotides is a condensation
reaction that releases a stable molecule
called Pyrophosphate (PPi – the i stands
for inorganic). A proton (H+) is also released.
- A Phosphodiester bond is formed between the
5’ Phosphate group of one nucleotide and
3’ Hydroxyl group of another.
- DNA is always synthesized in the 5’ to 3’ direction.

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Enzymes involved
- Helicase unwinds the dsDNA (double stranded DNA) by breaking hydrogen bonds.
- Single-stranded DNA binding proteins (shown in grey below) bind to each strand of the DNA to
prevent the unwound strands from joining.
- Topoisomerase breaks and reforms the DNA’s Phosphate backbone ahead of the replication fork to
relieve pressure that results from supercoiling (because DNA is completely supercoiled). In short,
topoisomerase prevents supercoiling of DNA as strands unwind. It relieves the pressure around the
replication fork.
- Primase synthesizes a short RNA primer required for DNA polymerisation. Polymerase will bind to
this primer to start synthesis.
- DNA Polymerase (specifically DNA Polymerase III) synthesizes daughter strands from parental
strands (in the 5’ to 3’ direction). Phosphodiester bonds are formed.

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- In the leading strand, Primase synthesizes an RNA primer and DNA Polymerase binds and builds the
new strand from there.
- In the lagging strand, Primase
synthesizes an RNA primer and DNA
Polymerase binds and builds the first Okazaki
Fragment. Primase then synthesizes a new RNA
primer and DNA Polymerase builds the next
Okazaki Fragment. The process is
repeated several times.
- DNA ligase joins (ligates) the Okazaki Fragments
together to form the lagging strand. Phosphodiester bonds are formed.
- Everything happens simultaneously. Primase synthesizes RNA Primer to start DNA replication, DNA
Polymerase extends the strands forming the Okazaki fragment, and DNA Ligase joins the fragments.
- When the bond between the Phosphates is broken, the energy released is used to form the
Phosphodiester between the incoming nucleotides and the existing chain.
Proofreading DNA synthesis
- When DNA Polymerase is synthesizing the new strands, it can make mistakes while adding
nucleotides. To minimize these mistakes, DNA Polymerase also has a proofreading ability.
- DNA Polymerase edits the DNA by proofreading every newly added base.
- When an incorrect base is detected, it is removed so that the correct one can be added again. DNA
polymerase will both remove the wrong base and add the correct one.
- When we speak about DNA primase in the 5’ to 3’ direction, we say it has polymerase activity, as it
can only build new strands in this direction.
- When we speak about DNA Primase in the 3’ to 5’ direction, we say it has exonuclease activity, and
this is its proofreading ability.
- This makes sense, because if it is building the new strand in the 5’ to 3’ direction, in order to
proofread, it has to go back and proofread in the opposite direction, which is the 3’ to 5’ direction.
- DNA Polymerase will stall if an incorrect base is added. It will not add another base until the
incorrect base is removed and corrected. The stalling of DNA Polymerase after insertion of an
incorrect base allows the proofreading activity (3’ to 5’ exonuclease activity) to remove the
incorrect base.
- Proofreading activity is slower than polymerising activity.
- Incorrect base pairs are added once in every 104 – 105 dNTPs (deoxynucleotide triphosphates –
recall nitrogenous bases added to the 1’ carbon form nucleosides. Nucleosides combined with 1 or
more phosphates form nucleotides. Deoxynucleotide triphosphates include dATP – deoxyadenosine
triphosphate, dCTP, dGTP, dTTP – see table on page 12).
- This error margin is very small (1 error per every 10 000 to 1 per 100 000 dNTPs added).
- The exonuclease ability of DNA Polymerase increases the accuracy of DNA replication by 100 to
1000 times. Thus, the error margin gets even smaller, and an error only exists once in about every
10 billion nucleotides.
- Note that the 1 in 10 billion errors still exist, and this is because DNA Polymerase replaces an
incorrect nucleotide with another incorrect nucleotide, and this can cause genetic mutations and
cancers.
- DNA replication is semi-conservative and bi-directional (5’ to 3’ direction at leading strand and 5’ to
3’ in opposite direction at lagging strand – DNA is NEVER synthesized in the 3’ to 5’ direction).
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Transcription and classes of RNA – Week 1 Lecture 4
RNA Structure
- RNA is a polymer of purine (adenine and guanine) and pyrimidine (cytosine and uracil)
ribonucleotides linked by 3’5’ Phosphodiester bridges (Note that it is the bonds that are in the 3’5’
direction. RNA is still synthesized
overall from 5’ to 3’).
- The reason we call DNA “deoxyribose”
is because it has lost one oxygen. Note
the difference between DNA and RNA in
the diagram.
- RNA is single stranded, however,
occasionally the strand may pair with
complementary bases on the same strand.
This is called intra-strand hydrogen-bond base pairing. It is still
only a single molecule. Thus, RNA is single stranded with short
regions of complementary intra-strand base pairing which forms
complex shapes.
- In DNA, the number of Guanines would equal the number of
Cytosines and the number of Adenines would equal the number
Thymines due to complementary base pairing. Since RNA is
single-stranded, this will not be the case.
- RNA is hydrolysed by alkali because of the OH group on the 2’ carbon
of the pentose sugar and is thus unstable, easily degraded, and does not
survive very long on any surface, unlike DNA.
Transcription of DNA into RNA
- The synthesis of RNA is similar to DNA replication.
- We require a DNA template, Ribonucleotides (ATP, CTP, GTP and UTP), and RNA Polymerase (for
DNA replication we needed DNA Polymerase).
- Transcription produces a sequence of ribonucleotides complementary to the sequence of
deoxyribonucleotides in one strand of DNA.
- In DNA, the 5’ to 3’ strand is known as the coding/sense strand. This is the gene sequence used by
geneticists or given in journals. The 3’ to 5’ strand
Is called the template/antisense strand.
- Only the template/antisense strand (3’ to 5’) is
used for transcription (in DNA replication the
template/antisense strand provides continuous
replication, and the coding/sense strand provides
discontinuous replication).
- Therefore, the RNA produced will be complementary (have opposite base pairs) to the 3’ to 5’
strand and will be exactly the same as the 5’ to 3’ strand, except it will have Uracil instead of
Thymine.

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RNA Polymerase
- There are 3 classes of RNA Polymerase:
1) RNA Polymerase I which synthesizes rRNA (ribosomal RNA).
2) RNA Polymerase II which synthesizes mRNA (messenger RNA).
3) RNA Polymerase III which synthesizes tRNA (transfer RNA).
- RNA Polymerase is a very complex enzyme that consists of many subunits.
- Transcription factors bind to the promoter sequence and recruit RNA Polymerase. Thus, RNA
Polymerase attaches to transcription factors that have already bound at the promoter (revise The
Anatomy of a Gene on page 7). It forms part of a transcriptional complex that assembles on
promoters to regulate transcription of genes.
- Different promoters bind different transcription factors. The transcription factors bound determine
the strength with which RNA Polymerase will bind. Thus, RNA Polymerase binds to different
promoters with different strengths.
- There are some common consensus sequences for promoters (TATA and CAAT)
- Unlike DNA replication that requires Primase to synthesize a primer for DNA Polymerase to bind,
RNA Polymerase does not require a primer.
- RNA Polymerase catalyses a sugar-phosphate bond (Phosphodiester bond) between the 3’ OH of
one ribose nucleotide and the 5’ PO4 of another.
- RNA Polymerase has a speed of 20 nucleotides/second.
- Transcription starts at the transcription initiation (start) site which is found at the boundary of the
promoter and the beginning of the first exon.
- Transcription ends 10-35 bps (base pairs)
after a termination/polyadenylation signal is
reached.
- The section of the gene between the
transcription start site and the transcription
stop site is known as a transcription unit.
Process of Transcription (same as The Anatomy of a Gene, just a little more detail):
- Initiation
1) RNA Polymerase is bound to transcription factors on the promoter, and helicase unwinds DNA.
2) The new RNA strand is synthesized in the 5’ to 3’ direction and is complementary to the
template/antisense strand (3’ to 5’ DNA strand).
3) The first 8 or 9 nucleotides are linked together.
4) Initiation is now complete and the transcription factors will be released. The RNA Polymerase
leaves the promoter region and moves along the DNA template/antisense strand.

- Elongation
1) RNA Polymerase moves along the template/antisense DNA strand and helicase unwinds it,
forming an elongation bubble. As with DNA replication, topoisomerase prevents supercoiling of the
DNA as the strands unwind.
2) Nucleotides are added to the 3’ OH of the growing RNA chain in a specific sequence (A pairs with
U and C pairs with G) dictated by the template/antisense strand, and this is known as Watson-Crick
Pairing.

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3) The new RNA molecule (primary
transcript) forms a short RNA-DNA hybrid
molecule with the DNA template in the
elongation bubble.
4) The RNA strand (like DNA in DNA
replication) is synthesized in the 5’ to 3’
direction until a termination sequence is
reached.

- Termination
1) Termination is signalled by a
transcription termination sequence in the DNA sequence.
2) The newly synthesized RNA is cleaved and released from the transcription complex 10-30 bps
(base pairs) after this sequence.
Regulation of transcription/gene expression in Eukaryotes
Transcription is regulated in the following ways:
1) Chromatin Structure:
- Chromatin (DNA associated with histones) structure regulates how much RNA is produced
(regulates transcription).
Recall that histones have N-terminal tails that can be modified, and these modifications have great
effects on DNA.
- If the histones are acetylated, we have increased transcription.
- If the promoter is methylated, we have decreased transcription.
2) Signal Transduction:
- Signal transduction (the passing of messages from outside the cell into the cell) also regulates
transcription. Signal transduction results in the activation of transcription factors. Transcription
factors need to bind to the promoter of genes to regulate transcription.
3) Transcription itself:
- Transcription is regulated through transcription factors – if they are not present, transcription will
not occur.

- Control elements are present in the promoter (TATA and CAAT).

- Control regions called enhancers and silencers are present in the DNA sequence, and if bound,
enhancers would increase transcription and silencers would inhibit transcription.
- Cis acting (they are part of the same DNA strand) enhancer regions are binding sites for activator
proteins (which are proteins like transcription factors). The enhancer regions are usually far
upstream or downstream of the transcription start site.
- Activators bind to both the enhancer and RNA Polymerase II, forming a DNA loop in the process.
- The binding of activators recruits general transcription factors, which help from a transcription
initiation complex with RNA Polymerase II, and we get increased transcription/gene expression of
that gene.
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- Cis acting (they are part of the same DNA strand) silencer regions can be found far upstream or
downstream of the transcription start site. Silencer regions are binding sites for repressor proteins
(which are proteins like transcription factors).
- The repressor proteins then binds to both transcription factors and RNA Polymerase II at the
promoter forming a DNA loop.
- The repressor proteins then inhibit the RNA Polymerase II and inhibits the binding transcription
factors and hence inhibit or silence transcription from that gene.

4) Regulation of mRNA concentration (how much transcription has occurred):

- RNA Processing. The processing of RNA regulates its half-life. If it has a short half-life, it does not
last very long, and very little protein will be produced. RNA with a longer half-life will survive for a
longer time and more protein will be produced from it.
- RNA silencing. RNA can be silenced by a small molecule called miRNA (micro-RNA), and it causes
degradation of mRNA (messenger RNA) that it is complementary to.

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Classes of RNA
Transcription is the production of ANY RNA, not just mRNA. All RNA is produced through transcription.
Coding RNA:
- mRNA – Messenger RNA – serves as a template for DNA synthesis.
Non-coding RNA:
- tRNA – Transfer RNA – Adaptor molecule for translation of RNA. Binds amino acids.
- rRNA – Ribosomal RNA – Contributes to the formation of ribosomes.
- snRNA – Small Nuclear RNA – Forms part of spliceosomes, which splice out introns.
- snoRNA – Small Nucleolar RNA – Processes rRNA (ribosomal RNA) in the nucleolus.
- miRNA – Micro RNA – Regulates gene expression.
mRNA Processing
- In mammalian cells, mRNA is transcribed as a precursor molecule called pre-mRNA. This is simply
mRNA with both introns and exons.
- The pre-mRNA is modified.
- The 5’ end is capped. 7methylguanosine triphosphate is attached to it. This allows recognition of
the ribosome, processing of the pre-mRNA and for it to be exported out of the nucleus.
- The 3’ hydroxyl end is polyadenylated (20-250 ATP’s are attached to it). This helps to regulate the
half-life of mRNA. The longer the polyadenylated tail, the longer the mRNA will survive.
- mRNA is then cleaved and spliced to remove introns. This occurs inside the nucleus on particles
called spliceosomes by snRNA (small nuclear RNA) and associated proteins forming snRNPS (small
nuclear ribonuclear proteins). Spicing occurs at spice junctions (intron/exon boundaries).
- We have sequence conservation (sequences that are usually the same and thus easily recognised)
at intron/exon boundaries – The end of an intron may have an AG and the beginning of the
following intron may have GU.

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Alternative Splicing
- By splicing at different points instead of all the intron/exon boundaries, we can derive numerous
mature mRNA’s from a single pre-mRNA.
- This is how humans only have about 22 000 genes but are the most complex organisms. One gene
has many functions due to alternative splicing. Other organisms have far more genes than humans
but are far less complex. The number of genes in an organism is not an indication of complexity.

tRNA
- tRNA is about 75 nucleotides long.
- The primary sequence undergoes folding and intra-strand base
pairing. This forms a cloverleaf structure.
- tRNA has 4 arms:
1) Anticodon arm – Binds to codon on mRNA.
2) Acceptor arm – Has ACC and binds a specific amino acid
depending on the anticodon of the anticodon arm.
3) D arm – Has the base dihydrouridine.
4) TΨC arm (read as T Pseudo C arm) – Has T, pseudouridine
and C.

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rRNA
- Ribosomal RNA (rRNA) is transcribed as a long
precursor that is highly methylated. All uridine within
it is converted to pseudouridine.
- Large precursor rRNA’s are processed in the nucleolus
by snoRNA (small nucleolar RNA) associated with
proteins to form components for ribosomal subunits.
The intervening sequences (shown in grey) are cleaved
out and we are left with 3 different sizes of rRNA –
28S, 18S and 5.8S.
- A 5S rRNA is transcribed by RNA Polymerase III from a
separate gene cluster (it is made elsewhere).
- S stands for Svedburg unit and is a measurement of
size.
- The 18S rRNA is incorporated into the small ribosomal subunit.
- The 5S, 5.8S and 28S rRNA’s are incorporated into the large ribosomal subunit.
The Ribosome
- The ribosome is cytoplasmic nucleoprotein (it consists of nucleotides and proteins or ribonucleic
acids).
- It is the machinery for the translation of proteins from the mRNA template.
- The ribosome is made up of two major subunits – A 40S and a 60S subunit.

END OF WEEK 1

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The RNA Interference (RNAi) Pathway and miRNA – Week 2 Lecture 1
RNA interference Pathway
- RNAi is a biological process and is a sequence-specific (homology-dependent) gene silencing
pathway.
- It is initiated by double-stranded RNA (dsRNA).
- RNAi is a method of gene regulation that works by silencing a gene.
- RNAi has 3 pathways:
1) microRNA (miRNA) pathway – This is the only important one as it is the only one found in
humans.
2) Piwi-interacting RNA pathway
3) Endogenous small interfering RNA pathway.
- RNAi is present in most multicellular eukaryotes, some unicellular eukaryotes but not in
prokaryotes.
- Gene expression is responsible for every aspect of a cell’s life (growth, division, death etc.) and
since RNAi is a gene regulator it regulates all these processes. Examples include haematopoiesis
(formation of blood cellular components), differentiation, tumour suppression, and developmental
timing.
- Antiviral defence and transposon silencing are also regulated by RNAi but are regulated by the Piwi
and Endogenous pathways and are thus not present in humans.
- RNAi was discovered in 1998 by Andrew Fire and Craig C. Mello.
- microRNA (miRNA) was discovered in 2001.
miRNA Pathway
- miRNA is first transcribed (from RNA Polymerase II promoters) like any other gene and is first
expressed as large RNA called pri-miRNA (primary microRNA).
- Since mRNA (messenger RNA) is also transcribed from RNA Polymerase II, miRNA looks very similar
to mRNA. It has a 5’ cap and a 3’ polyadenylated tail. They are both large sequences.
- These large structures fold up on themselves to form hairpin loops/stem loops. This is because at
physiological temperatures and physiological pH single-stranded RNA’s will fold up into secondary
structures. tRNA is an example of this – it folds into the cloverleaf structure. The same is true of any
RNA. They all fold into a secondary structure.
- Pri-miRNA folds into hairpin loops (stem loops) and can have a single (monocistronic) or multiple
(polycistronic) hairpin loops.
miRNA Production
- miRNA is transcribed from RNA Polymerase II promoters, which is recognised by RNA Polymerase II.
- Pri-miRNA is formed in the nucleus and folds into hairpin structures.
- Within the nucleus, pri-mRNA are cleaved to release the hairpin loops (that are each 70-80
nucleotides long). The hairpin loops are called pre-miRNA (precursor microRNA). The processing of
pri-miRNA to pre-miRNA is done by a microprocessor complex consisting of Drosha and DGCR8
(DGCR8 is Drosha’s binding partner). DGCR8 has dsRNA binding properties, and thus binds to the
double-stranded pri-mRNA, and DROSHA cleaves it.
- Pre-miRNA has a 2-nucleotide long overhang on the 3’ side. See here (the bottom strand):
2 nucleotide 3’
overhang →
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- Pre-miRNA’s are transported to the cytoplasm via a protein called exportin-5 (Exp-5 or XPO5) in an
energy-dependent manner. Exportin-5 binds to the pre-miRNA and carries it out of the nucleus and
uses energy to do so. Once exportin-5 releases the pre-miRNA in the cytoplasm, it heads back to
the cytoplasm to grab another one.
- In the cytoplasm, the pre-miRNA undergoes a second processing step to form the miRNA duplex.
This is done by Dicer and its dsRNA-binding partner TRBP. Dicer is an RNase (an enzyme that cleaves
RNA). TRBP binds to the pre-miRNA and Dicer cleaves it, forming the miRNA duplex. Dicer removes
the loop of the pre-miRNA to leave the double-stranded miRNA duplex. This duplex is 21-23 base
pairs long.
- The miRNA duplex enters RISC (RNA Induced Silencing Complex), and one strand is removed. The
removed strand is called the passenger strand and the strand remaining is called the mature miRNA
or the guide. It is called the guide because it guides the complex to complementary mRNA
(messenger RNA) which will be silenced (this is why we say RNAi is sequence specific, because only
something that is complementary to the miRNA will be silenced).
- The most important component of RISC is something called Ago-2.
- If the miRNA is a perfect match (every base in the guide has a corresponding complementary base
in the target mRNA) for its target mRNA, degradation will occur (Ago-2 will cleave the
Phosphodiester bond in mRNA). If it is an imperfect match (only some bases match), the miRNA will
inhibit translation of its target mRNA (because Ago-2 cannot cleave the mRNA). Either way, the
mRNA will be silenced, the protein will not be formed, and the gene will be silenced.
Some important points
- We know that all mRNA is transcribed from DNA.
- DNA has coding portions and non-coding portions.
- mRNA is a protein coding RNA, and thus is the only RNA transcribed from the coding portion of
DNA. All other RNA’s (including miRNA) are transcribed from the non-coding portions of DNA.
- Drosha and Dicer both cleave the RNA and are called RNase III enzymes. When they cleave the RNA,
they both end up leaving 2 nucleotide long 3’ overhangs.

5’ Cap Polyadenylated tail

 2 nucleotide 3’ overhang by Dicer

2 nucleotide 3’ overhang by Drosha↑

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miRNA Dysregulation
The miRNA pathway is part of normal human funtioning and can be very beneficial. When it is dysregulted,
however, it can cause severe problems such as cancer. An example of this is Chronic Lymphocytic
Leukemia. When a 30 kb (kilobases) DNA deletion occurs on Chromosome 13, miR-15 and miR-16
(microRNA 15 and 16) are affected and this causes the cancer. This is because once the DNA portion that
codes for these miRNA’s are deleted, they can no longer silence their targets. If their targets are not
silenced, we get over-expression of cell proliferating and cell division markers, thus leading to more cell
proliferation and growth, ultimately leading to cancer. Numerous cancers work in a similar way. Even
though miRNA may not cause the cancer, it is still very important in the development of the cancer. We
will focus specifically on cell proliferation and cell division.
The relationship between miRNA and cancer
There are two ways through which cancer can be caused due to miRNA dysregulation:
1) Downregulation of miRNA.
A decrease in the expression of a miRNA targeting an oncogene leads to increased expression of the
oncogene .
- Through mutation (a miRNA’s promoter might be mutated), less miRNA can be produced or though
deletion of DNA as we saw in Chronic Lymphocitic Leukemia less or no miRNA may be produced.
For whatever reason, less pri-miRNA is produced.
- We now have less pri-miRNA produced, which leads to less pre-mRNA produced, which leads to less
miRNA duplex produced, which leads to less entry into RISC and fewer guides being produced.
- This means that there will be less targeting of the gene that is the target of the miRNA.
- If that gene happens to be an oncogene (genes which stimulate cell growth and division) we will
have less silencing, which means more expression of that gene. If the miRNA is downregulated, its
target (such as an oncogene) is upregulated.
- Since the oncogene is not silenced, we have increased cell growth and proliferation which leads to
cancer.
- miR-15 and miR-16 are freuently downregulated in B-cell Chronic Lymphocytic Leukemia.
- If the downregulation of a miRNA causes cancer, its target must be an oncogene.

2) Upregulation of miRNA.
An increase in the expression of a miRNA targeting a tumour suppressor leads to decreased expression of
the tumour suppressor.
- Through mutation (a miRNA’s promoter might be mutated) or duplication of DNA, more miRNA can
be produced. For whatever reason, we have more pri-miRNA produced.
- More pri-miRNA = more pre-miRNA = more miRNA duplex = more acivation of RISC = more
targeting.
- If the target happens to be a tumour supressor (suppress tumours by inhibiting growth or cell
division), the tumour suppressor will be silenced. We say the tumour supressor is downregulated.
- If the tumor suppressor is silenced, it can no longer downregulate cell growth and cell division.
- Since the tumour suppressor is silenced, we have increased cell growth and proliferation which
leads to cancer.
- miR-17~92 is frequenlty upregulated in B cell lymphomas.
- If upregulation of a miRNA causes the cancer, its target must be a tumour suppressor.
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Viruses and miRNA
Viruses have been shown to encode their own miRNA (miRNA found in their own genome) in humans and
exploit the RNAi and miRNA pathway. Viruses are very good at exploting the cell machinery of their host.
They can exploit the transcription and translation of a cell they infect. Viruses can hijack the miRNA
pathway in 2 ways:
1) They target the host’s genes.
- Viruses have been shown to inhibit cellular factors involved in innate or adaptive immunity. They
can encode miRNA’s that downregulate the natural killer cell ligand MICB (involved in the immune
response). Viruses that do this include Human Cytomeglovirus, Kaposi’s Sarcoma-associated
Herpesvirus, and Epstein-Barr Virus.
- Epstein-Barr virus encoded miRNA also downregulates the pro-apoptotic (apoptosis is programmed
cell death – cells are programmed to die when they are infected) protein PUMA.
2) They target their own genes.
- Viruses can encode miRNA that downregulates the expression of viral proteins. What this means is
that viruses such as Herpes Simplex Virus 1 encodes miRNA that target early transactivators such as
ICP0 and ICP4, which are needed for the virus to begin reproduction. This causes the virus to stay
latent and not replicate. In this latent state, it is hidden and the immune system cannot recognise
and eliminate it.
- When you are healthy and your immune system is strong, the virus stays hidden. When you
become sick or stressed and your immune system is compromised, the virus turns off its miRNA and
begins reproduction to infect you.
Exploting the RNAi pathway to our advantage
Exploting the pathway can be very useful to silence any gene you want silenced. In theory, you would
produce an miRNA complementary to the gene you want silenced. There are 2 systems to do this:
1) Synthetic systems:
- You could produce a sequence to mimic miRNA that looks like a pri-miRNA, a pre-miRNA or a
miRNA duplex and slot it in to the miRNA pathway. Due the cost, smaller sequences are better as
they are cheaper, and thus miRNA duplexes are usually produced. It would then be processed to its
guide and silence its target. You would have produced the miRNA with a specific target in mind, and
thus it would be a perfect match for its mRNA target. This would cause degradation of the mRNA.
You would not have the case where you have an imperfect match and translation is suppressed.
2) Expressed systems:
- You could produce a DNA sequence that will be transcribed in the cell into an RNA. This RNA would
then be the mimic and silence the target gene.

Exploiting the RNAi pathway is used in functional genomics where a gene is silenced and the effect is
observed. This tells us what that gene is resposible for. It is also used in gene therapy where nucleic acid
sequences like these mimics are produced to bring about a therpeutic effect, such as a guide against a
gene that belongs to a virus to eliminate the virus or a gene involved in cancer development to suppress
the cancer.

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DNA Techniques using Hybridisation: PCT, RT-Polymerase Chain Reaction and FISH –
Week 2 Lecture 2
Hybridisation is simply when the 2 complementary sequences of DNA come together – they hybridise and
form Hydrogen bonds. When DNA is heated, it will denature and separate. When it is cooled, it will join,
and we call this hybridisation. The interaction is maintained through 3 Hydrogen bonds between G and C
and 2 Hydrogen bonds between A and T/U.
Hybridisation is the establishment of a sequence-specific interaction between two complementary
nucleic acid sequences.

First technique: FISH – Fluorescence in situ Hybridisation

- Probes are simply pieces (fragments) of DNA or RNA manufactured in a lab. They can be labelled
fluorescently or radiocatively. Fluorescent probes have a bright colour attached to them so that we
can keep track of the probe when using it.
- Locus specific probes bind to a particular chromosomal region. This is useful when trying to
determine on which chromosome a particular gene is located.
- As the F in FISH suggests, FISH makes use of fluorescent probes.
- In Situ means in its original place.
- FISH is a molecular cytogenic (at the chromosome level) technique that uses fluorescent signals
and nucleic hybridisation to detect a sequence of interest.
- Essentially, FISH is a technique where we make a specific fragment of DNA in a lab, and then insert
it into a DNA sample to see if it binds. If it binds to the DNA sequence, we know that a specific DNA
sequence is present.
- FISH can be used for the identification of presence and location of nucleic acids within metaphase
chromosomes, interphase nuclei, fixed tissues and cells in culture.
- FISH is used in conjunction with flourescence microscopy (to see the results). If the fluorescent
probe hybridises to the DNA sample, the fluorescent probe-DNA sequence can be studied under a
fluorescent microscope. Note that it will only bind if the probe and sample sequence have high
sequence complementarity.
- There are different types of samples: formalin-fixed parrafin embedded tissues or fixed cell
suspension.

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The process of FISH:

a) A probe and sample are prepared.

b) The probe is labelled (very important step)

c) The probe and sample are dentatured (to make

them single-stranded so they can bind).

d) The probe is hybridised to the sample.

e) The sample is imaged.

- A probe is designed against a a DNA sequence of interest.
- It is then labelled through:
1) Direct labelling using flourophores, which are fluorescent molecules
2) Indirect labelling using haptens, which are small molecules against which a fluorescently labelled
antibody can be raised. The probe is essentially flourescenlty labelled, but with extra steps.
- Samples are prepared on glass slides.
- The sample is imaged by fluorescence microscopy.
- If a signal is observed, the probe is present and so is the sequence of interest.
- If there is no signal observed, it means the probe did not hybridise, which means the probe is
absent and so is the sequence of interest.
- The position (on which chromosome) can be determined through FISH, as the name suggests (in
situ).
We can perform FISH when the sample is in interphase or metaphase.
- Interphase:
DNA is less condensed and not duplicated.
The results of FISH perfomed during interphase appear as depicted →

- Metaphase:
Metaphase is induced.
DNA is duplicated in metaphase and exists as sister chromatids.
The DNA is highly condensed in metaphase.
The results of FISH performed during metaphase appear as depicted →

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1) Preparing a Probe
There are many ways to prepare a probe, but we focus on Nick translation, which is where we make nicks
or cuts in DNA. You then use nuceotides that are labelled to fill in the gaps and thus your probe ends up
being labelled.
- Begin with a double stranded probe.
- Introduce an enzyme called DNase I which
will randomly cut the DNA (make nicks).
- Introduce DNA Polymerase to fill in the
nicks with modified dNTPs
(deoxynucleotide triphosphates). DNA
Polymerase extends chains (5’ to 3’) from
nicks with modified dNTPs.
- Whne the probe gets filled, it incoporates
these modified nucleotides, and the probe
thus gets fluorescently labelled.

2) Denature Probe and Sample

Once the probe and sample have been prepared, they need to both be heated to denature them.
Chemicals can also be used to denature them. This is to make them both single stranded.
3) Hybridisation
The single-stranded DNA sample and fluorescently labelled probe must now be hybridised together.
- The denatured probe is applied to the slide/tissue.
- The slide is incubated to allow hybridisation.
- A probe that has not hybridised must be washed off.
4) Image sample
The sample must now be imaged by fluorescence microscopy.
- A laser is used to excite the flourophores (a particle which has the ability to absorb light at a certain
wavelength and then emit that light at another wavelength), which then emits the fluorescent
signal.
- Fluorescent hybridisation signals are then detected.
Applications of FISH
- Identification of chromosomal abnormalities.
- Helps with gene mapping (localisation of gene in the genome – chromosome location), analysis of
chromosome structural aberrations (things away from the norm), toxicological studies, and ploidy
determination.
- Cancer diagnostics – deletion of tumour suppressors, duplication of an oncogene, and translocation
of two genes.
- Genetic abnormality testing, such as monosomy (being one chromosome short) or trisomy (havine
once extra chromosome).

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Examples
You have performed FISH using a red and a green fluorophore to identify 2 sets of genes very close to each
other on the chromosome.
- Seeing a yellow signal is normal because the genes are so close to each other. The red and green
signal are super-imposed on each other forming a yellow signal. Two yellow signals will be shown
because humans are normally diploid.
- Polyploidy is having more than 2 sets of chromosomes, such as being triploid (having 69
chromosomes).
- Aneuploidy is having an extra chromosome or have a chromosome less. Down’s Syndrome is when
a person has 2 of each chromosome (as is normal), except chromosome 21, of which they have 3.

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Second technique: PCR – Polymerase Chain Reaction
PCR is divided in 3 steps:
1) Denaturation (950C):
- Double-stranded DNA is heated to 95 degrees to disrupt
hydrogen bonds between complementary bases to form
single-stranded DNA.

2) Annealing/ Hybridisation (~55oC):

- Primer (short fragments of DNA complementary to target
sequence) anneals the single-stranded DNA.
- The difference between this and FISH is that here, 2 primers
on each side of the region you are trying to amplify are
needed, and they are not fluorescently labelled.
- See the diagram alongside. One primer is needed to anneal
to the one DNA strand upstream and one primer is needed
to anneal to the other strand downstream.

3) Exension/ Elongation (68-72oC)

- Polymerase (as stated by P in PCR) extends primers by
adding dNTPs in a template dependent fashion, forming
new strands of DNA in both directions.

These steps are repeated 30 to 50 times to get an exponential amplification of the products. It is hence a
chain reaction, as stated in the CR of PCR.

Components of a PCR reaction

- Enzyme – Thermostable DNA Polymerase
- Buffers and MgCl2 – Make the environment condusive for the PCR to occur by providing the correct
ions to sustain the reaction and for the enzyme to work optimally.
- Primers – 2 primers that hybridise at each end of the fragment to be amplified.
- dNTPs (deoxynucleotide triphosphates) – These are the bases that DNA Polymerase will add to the
growing strand of DNA.
- Template DNA.

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PCR Automation
- PCR is now fully automated. It has thermal cyclers that do the temperature changes automatically.
- Most enzymes (such as DNA Polymerase) denature at high temperatures. Therefore after each
cycle, new enzyme must be added and this causes problems.
- A major breakthrough for PCR was the discovery of Thermostable DNA Polymerases. These are DNA
Polymerases found in bacteria called Extremeophiles that are found in places that have high
temperatures, such as thermal vents on the ocean floor or hot springs. The Polymerase is thus used
to hot temperatures and does not denature at high temperatures, making it useful to PCR.
PCR Mechanism
PCR yields a great amount of product.
- If you know the location of the primers, you can
determine the size of the PCR product since it will
be from the beginning of one primer till the end of
the other primer.
- If you start with one double-stranded DNA
sequence at the beginning of the reaction, you
have 2 dsDNA at the end of the first cycle.
Remember that this is repeated 30-50 times.
- In the second cycle, you are starting with 2 dsDNA, and you will
yield 4 dsDNA at the end of the cycle.
- This continues for 30 times or more, doubling at each cycle.
- This is what is meant by exponential amplication. At the 30th
cycle, we have over 1 billion copies.
- This is very useful for crime scenes, where small amounts of
DNA can be amplified.
- Covid-19 tests are PCR tests.
A formula can be derived to calculate number of copies produced.
𝑦 = 𝑎 × 2𝑥 where:
- 𝑦 is the copy number produced.
- 𝑎 is the starting copy number.
- 𝑥 is the number of cycles completed.

Detecting PCR Products

DNA is ultimately the end product of PCR, but it is not visible to the naked eye. A process called Gel
electrophoresis is used. This is the movement of charged particles in a fluid under the influence of an
electric field.
- PCR products are placed in a buffer in a gel.
- An electric current is passed through, and cations migrate toward the cathode and anions toward
the anode.
- DNA is negatively charged due to its Phosphate group, and will migrate from the negative to the
positive electrode.
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Agarose Gel Electrophoresis
- Not all DNA migrates at the same rate.
- Larger DNA molecules do not go through
the matrix of the gel as quickly as smaller
DNA fragments.
- Thus, when we load pieces of DNA on an
Agarose gel, we can effectively separate
DNA based on size, with the smallest being
furthest away from the wells where you
loaded them, and the largest being closer.
How do we get different sizes of DNA in a PCR
reaction?
- Where primers bind determine the size of
the PCR product.
- Lets say the first primer binds to bases 100
to 119 on the one strand and the other
primer binds to bases 481 to 500 on the other.

- Effectively, the product will be from 100 to 500, which is 400 bps long. This Polymerase, however,
tends to add one extra base, and thus 401. This extra base is usually an A added at the tail – recall
the Polyadenylated tail added by RNA Polymerase II.
- If you want to check that your PCR went well, run a sample of the completed product on an
Agarose gel under electrophoretic conditions, and with the help of a special dye that stains DNA,
you can visualise your PCR products under UV light and make sure that the size is correct.

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PCR Products
On the left we have a DNA ladder consisting of various fragments of DNA length. This helps determine size
of PCR prodcuts.

Applications of PCR
It is argued that PCR is the most versatile molecular tool to date.
- Detection of heriditary diseases.
- Forensic DNA detection.
- Identification of genetic fingerprints (paternity testing, forensic testing).
- Cloning of genes.
- Identification of transgenic plants.
- Sequencing.

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Third technique: RT-PCR – Reverse Transcriptase Polymerase Chain Reaction
- This uses RNA as a starting template instead of DNA, as is done with PCR.
- Thus, an extra step is required to convert the RNA to cDNA (complementary DNA) and this is done
with the help of an enzyme called Reverse Transcriptase.
- The product is double-stranded cDNA.
- This is useful when you need to determine the abundence of specific RNA transcripts present in a
cell or tissue or to measure gene expression.
- Note that the presence of a gene does not mean that it is being transcribed, and so measuring the
amount of specific RNA is a better indicator of whether the gene is expressed.
- There is another PCR method called Real-Time PCR, and it is different to RT-PCR. The abbreviation
for Real-Time PCR is Q-PCR. Q-PCR uses flourescence to monitor the amplification reaction in real
time.
Restriction Enzymes
Restriction enzymes cut DNA at specific sequences and are called Restriction Endonucleases.
- Restriction Enzymes/ Restriction Endonucleases act as molecular scissors.
- They each recognise a different sequence and only cut the DNA where that sequence is present.
An example is EcoRI (the I is a Roman
numeral for 1):
- EcoRI has cut the DNA at the
recognition sequence shown.
- Results in two fragments of DNA
with sticky ends.
- Look at how EcoRI has cut. It has
not done so cleanly between the
six bases. EcoRI left a TTAA tail on
the left and a complementary
AATT tail on the right, and these
are known as sticky ends.
- Not every enzyme cuts with sticky ends. Some cut and leave no tail.
- There are many Restriction Enzymes – some leave sticky ends and some leave blunt ends.

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Structure of Amino Acids and Translation – Week 2 Lecture 3
Structure of an Amino Acid
- Amino acids are organic molecule.
- It consists of a Carbon attached to an Amino group (H3N+), a
Hydrogen (H), a Carboxyl group (COO), and a variable side
chain (R).
- The variable side chain is what gives an amino acid its
specificity because each amino acid has a different variable side chain.
- Glycine is the simplest amino acid because its variable side chain is just one Hydrogen.
- The way in which variable sidechains interact with each other determines how a protein folds, and
how a protein folds determines its function.
Hydrophobic (water-hating) Amino Acids:
- Hydrophobic amino acids are non-polar.
- Hydrophobic Amino Acids hate water and thus tend to be found
away from the outside of the protein that encounters water/liquids.
- Hydrophobic amino acids are the largest amino acids.
- There are two types of Hydrophobic Amino Acids:

1) Aliphatic Hydrophobic Amino Acids:

These are called Aliphatic because they consist of a variable side
chain consisting of Carbon and Hydrogen.
You do not need to know the structure of each amino acid →

2) Aromatic Hydrophobic Amino Acids:

These are called Aromatic because they have a benzene ring in their
variable side chains.
You do not need to know the structure of each amino acid →
Hydrophilic (water-loving) Amino Acids:
- Hydrophilic amino acids are charged.
- Hydrophilic amino acids tend to be found on the surface of proteins
as they can interact with water or fluid.
- There are three types of Hydrophilic Amino Acids. Two of these are
charged:

1) Basic Hydrophilic Amino Acids:

These have a positively charged variable side chain.
An NH2 is found in their side chain.
You do not need to know the structure of each amino acid →

2) Acidic Hydrophilic Amino Acids:

These have a negatively charged variable side chain.
A COO is found in their side chain.
You do not need to know the structure of each amino acid →
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- The last type of Hydrophilic Amino Acids is Polar:

3) Polar Hydrophilic Amino Acids:

These amino acids are uncharged.
There are two types of Polar Hydrophilic Amino Acids:
1) Polar Hydrophilic Amino Acids containing an Amide group.
H2N – C = O
2) Polar Hydrophilic Amino Acids containing a Hydroxyl Group.
OH

Other Amino Acids:

Sulphur containing R groups
- These amino acids have Sulphur in their variable side chains.

Proline
- This amino acid has a cyclic sidechain, but its not a Benzene ring so it
is not an Aromatic Amino Acid.
- This amino acid’s side chain is made of only Carbons and Hydrogens,
which normally would make it an Aliphatic Amino Acid, but it is in a
ring and thus cannot be Aliphatic.

Peptide Bond Formation

- Two amino acids are linked together through peptide bond formation.
- The peptide bond is formed through Dehydration Synthesis, and we have loss of a water molecule.
- Take note of the C = O and N – H; it will be useful later.

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A sequence of amino acids:
- Note all the different variable side chains. It is the interaction of these side chains with each other
which determines how a protein folds, and how a protein folds determines its function.
- Proteins are synthesized from the N-terminal to the C-terminal (carboxy terminal).
- Note that Lysine (Lys) is positively charged, and Glutamic acid (Glu) is negatively charged. They will
attract each other, and this affects structure.

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The Genetic Code
- The genetic code is the specific sequence of nucleotides in mRNA that determines what amino acid
is required through complementary base pairing with the anticodons on tRNA’s. Hence, the genetic
code determines polypeptide amino acid sequence.
- The nucleotides of mRNA are organized into code words called codons.
- There are 20 different amino acids in the human body, and if each mRNA codes for a specific amino
acid, there must be at least 20 mRNA codons as well.
- mRNA is made up of 4 different nucleotides (A, U, G, and C).
- Each codon is made of 3 nucleotides and is called a triplet code (such as AUG), and thus means that
there are 64 possible mRNAs. Think about it. If codons were made of 2 nucleotides, we would only
have 16 possible combinations, but there are 20 amino acids, and so we need more than 16
codons.
- UAA, UGA and UAG do not code for anything. Recall that they are stop codons (chain terminators).
- AUG codes for Methionine and recall that it is a start codon.
- There are thus 61 codons that code for 20 amino acids, thus making the genetic code degenerate
(each amino acid is coded for by 2 or more different codons).
- Codons are unambiguous – the same codon will always code for the same amino acid.
- Codons are non-overlapping, non-punctuated, and universal.
- This table showing mRNA codons will always be provided in exams if it is needed:

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Translation of RNA into amino acid sequence of protein.
- Translation is protein synthesis mediated by the interplay of mRNA, tRNA, activating enzymes,
protein factors and ribosomes attached to endoplasmic reticulum (ER) or free in the cytoplasm.
- Translation begins with the addition of COOH (carboxyl) group of an amino acid to the 3’ end of a
specific tRNA.
- The amino acid is added to the tRNA by an enzyme called Aminoacyl tRNA Synthetase. There are 20
amino acids, and each one has its own Aminoacyl tRNA Synthetase. There are thus 20 Aminoacyl
tRNA Synthetases. Each one of these enzymes recognise all tRNA’s for its specific amino acid.
Ribosomes

- Recall that a ribosome has a large and small subunit. When these are together, we get three sites
formed, namely and A site, a P site, and an E site. See below:

The process of Translation:

Like transcription, translation also has three stages – Initiation, Elongation, and Termination.
1) Initiation
- The 5’ end of mRNA binds to the small ribosomal
subunit.
- The ribosome moves along the mRNA until the
initiator codon (AUG) is in the P site.
- A tRNA anticodon complementary to AUG binds (this
is UAC).
- Methionine is the amino acid coded for by the AUG
codon or UAC anticodon.
- Methionine is the starting amino acid of all proteins
but is sometimes removed after translation.
- Eukaryotic initiation factors (eIFs) regulate the
binding of the large ribosomal subunit to the small ribosomal subunit.

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2) Elongation
Elongation occurs in 3 steps.
Elongation Step 1
- After initiation, the A site of the ribosome was empty.
- Now, a tRNA with an anticodon complementary to the mRNA
codon (now in the A site) binds.
- This requires GTP (Guanine Triphosphate).
Elongation Step 2
- Elongation is the process by which amino acids are added one
after the other to from the chain of amino acids or polypeptide
of protein.
- A ribozyme called Peptidyl Transferase forms a peptide bond
between the amino acid in the A site (which was Methionine –
the initiatal amino acid) and the amino acid in the P site. These 2
amino acids (dipeptide) now bound to each other are on the
tRNA in the A site and the tRNA in the P site that had the
Methionine has no amino acid and is thus uncharged.
- This process is a dehydration reaction and results in the release
of water.
Elongation Step 3
- The large ribosomal subunit translocates (shifts) to the right,
and then the small ribosomal subunit translocates to the
right. This requires GTP.
- The dipeptide is in the P site.
- The tRNA without an amino acid is in the E site (Exit site) and
is released.
- The A site is empty.
- Eukaryotic elongation factors (eEFs) regulate this process.

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- The process continues.
- A tRNA complementary to the mRNA will bind in the A site.
- A peptide bond forms between the new amino acid in the A site and the and the existing dipeptide
in the P site, forming a tripeptide in the A site.
- Ribosome shifts to the right, and the tripeptide is in the P site and the uncharged tRNA with no
amino acid is in the E site and is released.
- A site is empty (has no tRNA) and a new corresponding tRNA will bind.
- This continues over and over.
3) Termination

- Termination occurs when a stop codon (UAG, UGA, UAA) appears in the A site.
- Stop codons do not code for an amino acid because no tRNAs recognise stop codons. tRNA does not
bind to stop codons.
- The stop codon is recognised by a release factor.
- The release factor binds to the stop codon and adds H 2O (water) to the end of the polypeptide
chain.
- The polypeptide is released from the P site, and ribosome subunits dissociate from each other.
- Remember that only uncharged tRNA’s with no amino acid exit through the E site.
- Eukaryotic release factors (eRFs) regulate this process.
Polyribosomes
- Multiple ribosomes read the same mRNA strand
at the same to produce many proteins at once.
- These polyribosomes (multiple ribosomes) can
be present in the cytoplasm or on the
endoplasmic reticulum (ER).
- If a protein is needed in the cytoplasm, it will be
made in the cytoplasm.
- If a protein is required to be secreted or act as a
membrane receptor or is present in the
lysosome, it will be made directly in the ER.

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A Summary of Transcription and Translation

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Processing of Proteins formed in the Endoplasmic Reticulum
- A short signal sequence on the newly formed peptide (the short sequence of amino acids still
growing) directs the mRNA and attached ribosome to ER.
- The ribosome remains in the cytoplasm on the ER and produces the growing polypeptide into the
ER (the growing polypeptide passes into the cisterna of the ER).
- The signal sequence initially remains attached to the receptor but is soon clipped off by an enzyme.
- As the protein synthesis continues, sugar or carbohydrate groups may be added to the protein.
- Once the protein is completed, it is released from the ribosome into inside the ER and folds into its
3-D conformation, and this is aided by molecular chaperones.
- Once in its mature form, vesicles containing the protein will bud off from the endoplasmic
reticulum. These are known as transport vesicles.
- Transmembrane proteins (proteins that are found on the membranes of cells) remain embedded in
the membrane – they are not released into the ER but remain on the ER membrane. This portion of
the ER membrane may be pinched off into a transport vesicle as well.
- The transport vesicles travel to the Golgi apparatus, where further processing and sorting of the
proteins occur.

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Processing of Proteins in the Golgi Apparatus
The Golgi Apparatus consists of stacked and flattened membranous sacs shaped like hollow dinner plates
called cisternae, associated with many tiny membranous vesicles.
- The transport vesicle travels from the ER through the cytoplasm to the Golgi apparatus and fuses
with the Golgi apparatus at its cis face.
- The proteins from the transport vesicles can now be found in the lumens of the Golgi apparatus.
- Newly formed vesicles become transport vesicles and they either become secretory vesicles,
membrane vesicles, or lysosomal vesicles.
- The major function of the Golgi apparatus is to modify, concentrate, and package the proteins
made at the rough ER.

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There are thousands of proteins in our bodies. They may be sorted into:

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Protein Structure and Post Translational Processing – Week 2 Lecture 4
Proteins are the most abundant organic molecule in
living systems. Recall that there are 20 different
amino acids and the only difference between each
of them is their variable side chain.

- The beginning of a protein is called the

amino end/terminus.
- The end of a protein is called the carboxyl
end/terminus.
- Proteins are produced as linear molecules.
When they fold, the hydrophobic (non-
polar) side chains move to the centre where
they will be away from water and the hydrophilic (polar) side chains move the outside where they
will encounter aqueous solutions.
- Peptides have 2 to 30 amino acids.
- Proteins are made of 50 to 1000 amino acids.

There are 4 levels of protein structure:

1) Primary structure:
- The sequence of amino acids in the polypeptide chain as determined by mRNA codons. Contains
peptide bonds.
- Ultimately, the unique sequence of any protein is determined by the gene that encodes the protein.
Any change in the gene sequence may lead to a different amino acid being added to the
polypeptide chain, causing a change in protein structure and function.

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2) Secondary structure
Recall that when amino acids bind, we get a N-H and C=O.

- Secondary structure results from the hydrogen-bonding between the N-H and C=O groups in the
polypeptide backbone.
- This causes folding and there are two regular folding patterns found as dictated by amino acids in
primary sequence – secondary structure.
- The two folding patterns are:

1) Secondary structure: α helix

- In α helix, a single polypeptide chain twists to form a rigid cylinder.
- Hydrogen bonds within the polypeptide chain are spaced four amino acids apart.

2) Secondary structure: β pleated sheet

- Rigid planar structures with hydrogen bonds between amino acids in adjacent strands form.
- β sheets can form either from neighbouring polypeptide chains that run in the same orientation
(parallel chains) or from a polypeptide chain that folds back and forth upon itself, with each section
of the chain running in the opposite direction of its immediate neighbours (antiparallel chains).

3) Tertiary structure
- Tertiary structure refers to the unique 3-D structure of a
polypeptide.
- The structure is caused by the chemical interactions
between amino acid side chains.
- These interactions include disulphide bonds, hydrogen
bonds, ionic bonds, hydrophobic interactions, and
disulphide bonds.
- Forms by secondary structures folding with each other.
- Tertiary structure is the highest level of organisation in
monomeric proteins (monomeric means molecules that can
combine with other to form polymers).

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4) Quaternary structure
- Quaternary structure describes the number and relative
positions of subunits in multimeric proteins (proteins composed
of several subunits).
- The image showing a quaternary protein has 4 subunits and is
called a tetramer:
- If the quaternary protein was composed of three subunits, it
would be called a trimer.

Summary

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Protein Domains
- A protein domain is a portion of a protein that is composed of 40-350 amino acids and has tertiary
structure of its own (meaning it is biologically active) and is independently folded.
- It is the modular unit from which many larger proteins are constructed.
- Different domains are often associated with different
functions.
- Small proteins have a single domain, and larger
proteins can contain as many as several dozen
domains, usually connected to each other by short,
relatively unstructured lengths of polypeptide chain.
- An example is the SH3 (SRC Homology-3) domain. SH3
domains are small domains of around 50 amino acids
residues that are involved in protein-protein
interactions. SH3 domains have a characteristic 3D
structure, as seen in the image alongside →
SH3 domains occur in a diverse range of proteins with
different functions, including adaptor proteins, phospholipases and myosins. MCK is a cytoplasmic
adaptor protein that contains multiple SH3 domains. It is involved in transducing signals from
growth factor receptor Tyrosine Kinase (recall from the first page that tyrosine kinase is involved in
cell signalling) to downstream signal recipients.
- Multiple domains can occur in a single protein, and these can be the same or different.

Post Translational Modification (PTM)

- PTM refers to the modification of proteins after they have been synthesized resulting in changes in
cellular functions, structure and can impact the interactions of proteins. We can call proteins
dynamic since they can be modified to elicit changes in cellular functions.
- The Human Proteome (which refers to the proteins that can be expressed by a cell, tissue or
organism) is more complex than the Human Genome (which refers to the complete set of genes of
an organism). This is because single genes can code for multiple proteins, and hence there are more
proteins expressed than genes. PTMs further increase this diversity.
- PTMs regulate localisation, activity and interactions with other cellular molecules.
- PTMs can occur at any time during the life cycle or biosynthesis of a protein:
1) PTMs can occur after translation to mediate proper folding of the protein. This can increase
stability of the protein or help localise the protein to a distinct cellular compartment.
2) PTMs can occur after protein folding and localisation. This can alter the biological activity of the
protein.
- PTM can occur through the addition of chemical groups. To be modified, proteins may undergo
phosphorylation, acetylation, hydroxylation and methylation.

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Phosphorylation
- Phosphorylation involves the addition of a phosphate group on serine, threonine or tyrosine
residues and is one of the most important and extensively studied PTM in both prokaryotes and
eukaryotes.
- The addition of a phosphate group can convert a previously uncharged pocket of protein into a
negatively charged and hydrophilic protein, thereby inducing conformational change in the protein.
- The transfer of the phosphate is facilitated by enzymes known as protein kinases, and enzymes that
help to remove the phosphate group are called Phosphorylases. Since phosphate groups can be
added or removed, we say that the proteins have reversable modification.
- An example is P53, a tumour suppressor protein used in cancer therapy that is activated by
phosphorylation of its N terminal (beginning terminal) by several kinases.
Methylation
- Methylation refers to the addition of a methyl group to Lysine or Argenine residues of a protein.
- Methylation involves the transfer of C (methyl groups) to Nitrogen or Oxygen to amino acid side
chains to make the protein more Hydrophobic. Methylation can neutralise a negative amino acid
charge when bound to Carboxylic Acids.
- Argenine can be methylated once or twice, while Lysine can be methylated once, twice, or thrice.
- Methylation is achieved by enzymes called methyl transferases.
- Methylation has been widely studied in histones, wherein histone methylation (on the N-terminal)
can lead to gene activation or repression, based on the residue that is methylated.
- P53, the tumour suppressor mentioned above can be methylated to regulate gene transcription.
- Histone methylation of Lysine and Argenine residues in the N-terminal tail of histone H3 can
influence chromatin organisation.
- When such methylation reactions occur within gene promotor regions, they can activate or repress
gene transcription, depending on their location.
Acetylation
- Acetylation regulates many diverse functions, including DNA recognition, protein-protein
interactions, and protein stability.
- Protein acetylation plays a particularly important role in chromatin remodelling and is associated
with the activation of gene transcription.
Glycosylation
- Glycosylation refers to the addition of saccharides (sugars) to proteins to produce proteoglycans in
the ER (endoplasmic reticulum), Golgi, or cytoplasm.
- They have critical roles in protein sorting, immune recognition, receptor binding, inflammation, and
pathogenicity (the property of causing disease).
- Glycosylation plays an important role in protein folding, conformation, distribution, stability, and
activity.
- Examples of Proteoglycans include the ABO blood group proteins on red blood cells and gp120
(glycoprotein 120) of HIV.

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Protein Homeostasis in the Cell – Week 2 Lecture 5
Autophagy

- Autophagy is the destruction of “large” cytoplasmic contents.

- During the process of autophagy, the cell produces a membrane which surrounds the proteins or
organelles that are going to be destroyed. This forms an Autophagosome.
- The Autophagosome combines with a Lysosome and the contents of the Autophagosome are
digested.
- This process is not very discriminating as autophagy can:
- Destroy organelles no longer required by the cell.
- Destroy large protein aggregates – this is when there has been oxidative damage to the cell and
many proteins have been damaged.
- Activated in starvation to produce nutrients for the cell. This why when you diet, you breakdown
muscle to feed your body.
We need a more specific and targeted process to destroy specific proteins:
- This can include abnormal proteins, normal proteins no longer required by the cell, or foreign
proteins such as those produced by viral infections within in the cell.
- Abnormal proteins can include mutant proteins, oxidised/denatured proteins or incorrectly folded
proteins.
Protein folding
- Protein folding is a complex process, and every cell has a process for correcting abnormal folding, as
it is very dangerous to have abnormally folded proteins in the cell. This is because you could have
hydrophobic proteins on the outside which can then interact with various membranes within the
cell and cause damage to the cell.
- You do not need to know the following diagram, just read it. Note that this process requires energy
in the form of ATP.

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Normal Protein Levels in the Cell
- There must be a balance between protein production and loss or destruction of proteins in a cell.
- Normal proteins must sometimes be destroyed as proteins within a cell may need to increase or
decrease depending on the requirements or response of a cell.
- Both the concentration and timing of the presence of proteins in a cell is important for cell
function.
- Some proteins are short lived, whist others’ life spans can vary.
- Proteins whose concentration needs to be controlled may include:
- Transcription factors – these need to be regulated so that they are present when the cell wishes to
transcribe genes and absent at other times so that the gene is kept silent.
- Cell cycle control proteins – these need to control the phases of the cell cycle.
- Inducible proteins (like some enzymes) – the more substrate there is, the more enzyme need to
be produced, and we need to control for that.
- DNA repair proteins – these should only be present when there is DNA breakage or a problem
with the DNA needs to be sorted out. Extra proteins should not be kept in the cell if they are not
required.
- NFkB signalling – will be discussed in immunology, but it is a signalling pathway that is critically
dependent on the breakdown of certain proteins.
The System that Destroys the Individual Protein: The Proteasome
- The name Proteasome is derived from Protease, an enzyme which breaks down proteins.
- The Proteosome is found in both the nucleus and the cytoplasm, and proteins can thus be broken
down on either of the places.
- The components of the Proteasome include:
- Core particle – contain catalytic (proteolytic) sites which do the actual breaking down of the
protein.
- Regulatory caps – found on each end and each made of a base and a lid. Regulatory caps protect
proteins that should not be destroyed from getting into the core particle. They act as “stoppers” on
each end which protect and limit the number of proteins that can get inside the catalytic sites.

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How does the Proteasome know which proteins to destroy?
- Answer: The Ubiquitin protein – derived from the word
“ubiquitous” meaning “found everywhere” or “widely
present”.
- A chain of ubiquitin molecules (polyubiquitin) is attached to
the targeted protein as a signal for destruction.
- Ubiquitin is always present in a cell and then can be used to
attach (as a chain of ubiquitin molecules) to a protein to
allow the Proteasome to recognise the protein for
destruction.
- The ubiquitins bind to the Regulatory cap of the Proteasome
and are released by the cap. It is not broken down and can
attach itself to another protein.
- The protein is unfolded and passes into the cylinder of the
proteasome, where the catalytic sites will digest the protein
into small peptides or even individual amino acids.
- Know how to draw this:

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Polyubiquitin Tagging
- This is the process through which ubiquitin is added to a protein.
- There are 3 enzymes in the cell important for this process:
- E1, which is the Ubiquitin-Activating Enzyme.
- E2.
- E3.
- The first step is with the Ubiquitin-Activating Enzyme (E1), which activates the ubiquitin. This uses
ATP but is not a Phosphorylation reaction. It uses the energy of ATP to change the energy state of
ubiquitin, and the ubiquitin is then bound to E1.
- Once the ubiquitin is bound to E1, the E1 associates with an E2 and E3 and the E1 transfers the
activated ubiquitin onto E2. The E1 then leaves the complex.
- The E2 (with ubiquitin bound) and E3 are now ready to receive the protein that needs to be tagged.
- The target protein is now bound to this ubiquitin ligase.
- The particular shape of the E3 will recognise the particular shape of the protein, as E3 provides a
specific binding site for the targeted protein. This holds the protein for this process to occur.
- The E3 ligases catalyse the transfer of Ubiquitin from E2 directly to a Lysine in the substrate protein.
- The ubiquitin is transferred from the E2 directly to the protein.
- This whole process is repeated several times until we get a polyubiquitin chain. The initial ubiquitin
is added directly to the protein. Subsequent ubiquitin molecules are added to the previous
ubiquitin molecule.

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Specificity of the Tagging Process
- The specificity of the Polyubiquitin Tagging process is achieved through having multiple versions of
the 3 enzymes required (E1, E2, and E3).
- In humans there are:
- 2 different versions of E1.
- +/- 60 versions of E2.
- +/- 600 versions of E3.
- If you combine a specific E1, a specific E2, and a specific E3, you get a pathway that is very specific
for a particular protein. Once the E2 and E3 are brought together, it will only recognise 1 particular
protein and no others.

Activation of this Pathway needs to be Specific and Controlled

- As stated previously, we have to control the
concentrations and the timing of certain
proteins in the cell with absolute split-second
timing. If not, control of the cell may be lost and
this is a very dangerous process.
- There are different ways in which you can
change either the protein or the E3 to recognise
each other.
- If you change the protein, then an E3 which is
already there can recognise it.
- If you change the E3 by modifying it in some way, it will be able to recognise the protein.

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How can the Protein be changed?
- The first way is through Phosphorylation. What the protein looks like changes when it has a
phosphate group added to it. Phosphorylation is an important way of changing proteins.
- The second way is removing an extra protein subunit or associated protein that is masking the
binding site for the E3 ligase.
- The third way is by cleaving off a part of the protein that is hiding the binding site.

How can the Ubiquitin Ligase be activated/change its recognition site for the protein?
- The first way is through Phosphorylation. Phosphorylation is an important way of changing proteins
(like E3) and allows them to change their function.
- The second way is through adding a ligand. This brings about a change in the conformation of the
E3.
- The third way is through adding a protein, which will change the conformation of the E3 and allow
it to recognise the protein that it needs to bind to.
Role of The Proteasome in the immune system
- Short pieces of foreign peptides (from viruses and bacteria) are displayed on the surface of cells as
part of the immune process.
- The proteasome is specially modified (by the use of a different cap and proteolytic enzymes), to
produce that specific length of peptide required.
Malfunctioning of The Proteasome Pathway
- Dysregulation of the proteasome can cause
neurodegenerative diseases such as Alzheimer’s disease
and Parkinson’s Disease.
- In Parkinson’s, PARK2 (which encodes the E3 ligase parkin)
mutations lead to familial Parkinson’s disease.
- In both Alzheimer’s and Parkinson’s disease, there is an
intracellular or intraneuronal accumulation of misfolded
proteins which would normally be broken down by the
proteasome system.

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Manipulation of The Proteasome Pathway
- Bortezomib is an anti-cancer drug. It blocks the proteasome and appears to cause increase
apoptosis (programmed cell death) and decreased proliferation of some types of cancer cells. It
blocks NFkB signalling (recall that this signalling relies on the breakdown of proteins, and if the
proteosome is blocked, proteins will not be broken down).
- Bortezomib combats these cancers in two ways:
1) Myelomas (cancer of plasma cells) and Pancreatic Carcinomas (tumour of the Pancreas) are
treated with Bortezomib. This is because these cancers rely on the NFkB pathway for signalling
and proliferation of the cells. Plasma cells normally use the NFkB pathway, and Myeloma which
is cancer of the plasma cells thus also uses the NFkB pathway. Inhibiting the NFkB pathway
therefore inhibits the proliferation of the Myeloma cells.
2) Because Myelomas come from plasma cells (which produce antibodies), they are also cells
which produce large numbers of proteins. In any cell that produces proteins, there is going to
be cases of proteins which have not folded correctly. If these misfolded proteins are allowed to
accumulate, it will cause damage to the cell and the cell will die. Since Bortezomib blocks the
proteasome, these misfolded antibody proteins will accumulate and damage the cancer cells.
Pancreatic Carcinomas are another type of cancer in which large amounts of protein are
produced, and Bortezomib will allow the accumulation of misfolded proteins which will damage
the cancer cells and allow them to die.

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Protein Analysis Tools – Week 3 Lecture 1
- Protein analysis tools and techniques enable one to separate proteins and subsequently identify
them.
- Separation and identification methods include:
- SDS-PAGE
- 2D-Gel Electrophoresis
- Isoelectric focusing (IEF)
- Western blot
- Protein purification of methods include chromatography.
- Insulin is a protein that is produced in a lab for patients who cannot make it themselves. It is
important to use analysis tools to ensure the correct protein (insulin) has been made and that it
contains no other proteins that could be harmful to people.
SDS-PAGE
- SDS-PAGE stands for Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis.
- A solid medium in the form of a gel is submerged in a buffer solution.
- The gel has wells on top in which a protein sample can be transferred using a pipette.
- Because the density of the protein sample is typically similar to that of the buffer solution, the
sample’s density is usually increased by mixing it with a loading dye enabling the sample to be
suspended in the wells of the gel.
- The gel enables us to see the migration of the protein across the gel upon applying an electric
current.
- The protein migrates from the cathode end (negative) to the anode end (positive) of the gel,
enabling the protein to migrate along the gel in the same direction.
-

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SDS:
- SDS (Sodium Dodecyl Sulphate) is an amphipathic detergent with an anionic tail and a lipophilic tail.
- SDS is used to denature and dissociate proteins from each other and confers a negative charge on
the protein, masking the protein’s intrinsic charge.
- SDS-treated proteins have similar mass to charge ratios and shapes.
PAGE:
- During PAGE (Polyacrylamide Gel Electrophoresis), the rate of protein migration is determined by
molecular weight, where all protein migrates from the cathode to the anode.
- PAGE is the support medium, and the gel has pores which are determined by the concentration of
the acrylamide used to prepare it. A lower concentration of acrylamide will mean larger pores and
vice versa.
- Larger molecules travel slower than smaller molecules across the gel.
- In a gel with uniform density, the relative migration distance of protein (Rf) is negatively
proportional to the log of its mass.
- Performing a gel with proteins of known and unknown molecular masses simultaneously allows one
to estimate the masses of the unknown proteins by plotting the relationship between Rf and the
log of its mass.

2D-Gel Electrophoresis
- The aim of 2D-Gel Electrophoresis to analyse complex protein mixtures from cells, tissues and other
biological samples.
- 2D-Gel Electrophoresis is a combination of Isoelectric Focusing (explained on next page) and SDS-
PAGE (explained above).
- This is done by separating and identifying proteins in two steps or two dimensions.

Step 1 – Isoelectric focusing (IEF):

Proteins are separated according to their isoelectric
points (the pH at which a particular molecule carries no
net electrical charge).

Step 2 – SDS-PAGE
Proteins are separated according to their mass.

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Isoelectric Focusing (IEF)
- Isoelectric focusing is used for the analysis of complex protein mixtures from cells.
- A solution with proteins of various molecular masses and different charges are separated according
to their isoelectric points, where upon the application of an electric current through the gel matrix,
the protein becomes stationary at the point where its net charge is 0.
- A pH gradient is applied onto a gel and an electric current is applied across the gel, making one end
more positive than the other.
- Proteins are charged at all pH values besides their isoelectric values (the pH at which a particular
molecule carries no net electric charge).
- Proteins migrate along the gel until they reach their isoelectric points, where they remain
stationary.
- Positively charged proteins are pulled towards the negative end of the gel and vice versa.
- The pH gradient in the gel is formed by the presence of ampholytes, which are complex mixtures of
synthetic polyamino-polycarboxylic acids.
- In Isoelectric focusing, proteins are separated because of their charge rather than their molecular
mass.

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Protein Purification: Chromatography
- Proteins must be purified to ensure that the highest
purity of protein is obtained.
- Pure protein does not have additional cell components,
or other protein and/or contaminants that can
contribute to the efficient functionality of the protein
of interest.
- Chromatography is a fundamental component of most,
if not all, recombinant protein purification.
- There are many types of chromatography, but the
basic principle is the same. A sample containing the
desired protein is applied to a solid matrix and allowed
to elute (remove an adsorbed substance by washing
with a solvent) through a porous plug where the eluate
(a solution obtained by elution) can be collected.
- Over time, the sample is applied to the matrix, and various fractions of the sample can be collected,
based on the type of chromatography used for the purification.
There are 3 main types of chromatography, based on the protein that is the basis of the purification:
1) Affinity-Based Chromatography
The solid matrix, which is usually composed of beads, is coated
with a molecule which has an affinity for the protein.
For example, the beads maybe coated with an antigen that has a
high affinity to the protein, therefore causing the protein to bind
to the beads, and not be eluted with the rest of the sample. The
protein can then be collected using a relevant solvent that breaks
the bond between the antigen and the protein of interest, and the
protein fragment can be collected.

2) Size-Exclusion Chromatography
The solid matrix traps the protein within pores in the solid matrix.
Chromatography beads with specific pore sizes can be purchased
to enable entrapment of the protein (of known size) of interest.

3) Ion-Exchange Chromatography
Ion-exchange chromatography facilitates binding of the protein of
interest to the solid matrix based on opposing charges.
For example, in the image alongside, the solid matrix composed of
beads is positively charged and binds the negatively charged
protein molecules.

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Pure Protein
- In lane 2 in the image below, an affinity purified protein (pure protein) is shown, whereas lane 1
shows the banding pattern for the total protein lysate, prior to purification.
- Once a protein has been purified, there are many ways in which it may be utilised, as shown below.

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Recombinant Proteins – Week 3 Lecture 2
Recombinant DNA (rDNA) is a DNA strand that is formed by 2 or more DNA sequences which are often
from different organisms. The resulting Recombinant DNA is put into a host cell where it is expressed into a
new protein, called a Recombinant Protein (rProtein).
- Proteins are the most abundant molecules of living organisms and they play an important role in
structural and functional organisation of the cell.
- Recombinant technology refers to the recombination of genetic material of one organism with
another in vitro (in the lab).
- Recombinant material is introduced into a host cell.
- Recombinant proteins (rProtein) result from the expression of recombinant DNA (rDNA) within
living cells.
- Once rDNA is inserted into bacteria, these bacteria will make protein based on the rDNA being
translated into rProtein. This occurs like normal gene expression, where DNA is transcribed into
mRNA, which is then translated into protein.
Cloning Expression Cassettes
- Cloning expression cassettes enables protein expression and is illustrated as follows:

- The gene responsible for the protein is isolated from the DNA of a human cell using restriction
enzymes.
- The same restriction enzymes are used to cut a piece of DNA from a plasmid of a bacterial cell in
the example above. This does not necessarily have to be a plasmid from a bacterial cell.
- The DNA cut from the human cell (the gene of interest) is then joined or ligated to the piece of DNA
from the plasmid (in this specific example) obtained from the bacterial cell.
- This leads to the development of a recombinant plasmid/vector which is then inserted into a
bacterial host cell which multiplies with the recombinant plasmid within it.
- Note that in this example plasmid was used as a vector. There are other vectors that can be used.

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Recombinant Protein Expression
- As mentioned, rProteins involve the introduction of rDNA into a host cell, where the cellular
machinery will be utilised to express rProtein.
- To achieve this, a process called Bacterial Transformation is performed when the host cell is of
bacterial origin and is referred to as Cell Transfection when a Eukaryotic cell is used as the host cell.
- Recall that cells have cell membranes which regulate entry and exit of molecules into and out of the
cell to protect the cell from foreign molecules which may harm the cell.
- Transformation and Transfection enable the cell membrane to become porous to allow the foreign
molecule (in this case rDNA) to enter into the cell.
- Transformation can be done through Electroporation (where the cell membrane is treated with an
electric current to reach sufficient permeability for the introduction of rDNA into the cell) or
chemically (by calcium salts and heat shock).
- Transfection can be done through Electroporation (same as above), Chemically (with calcium salts
which is an old method, or with Lipids which is a modern method), or through Microinjection.
- The overexpression of the protein lead to the protein being harvested adequately.
- Learn the following, paying particular attention to advantages and challenges of each:

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Protein Expression and Purification Summary
- A gene on interest is isolated.
- The isolated gene of interest is inserted into an expression vector, resulting in a recombinant vector
or recombinant DNA.
- The recombinant vector/recombinant DNA is transferred into a host cell through Transformation (if
the host cell is bacterial) or transfection (if the host cell is Eukaryotic).
- The cell containing the recombinant proteins are then identified and isolated, and those cells are
allowed to grow.
- The protein is then isolated and purified.

Cloning Vectors
- Cloning vectors have important features that enable them to
play their role as carries of DNA fragments/genes of interest.
- A plasmid vector called pBR322 is shown alongside. This is a
commonly used plasmid and is used as an example to
illustrate the required features of a cloning vector, which
are:

1) Origin of replication (the site where DNA replication is

initiated).

2) Marker genes for selection and/or screening.

Selectable Markers:
Only cells containing plasmid forms colonies.
An example of a selectable marker is antibiotic resistance. Cells that are resistant to antibiotics can
grow on media containing antibiotic, and this means that only the cell containing the plasmid will
form colonies, because if the cells do not contain the plasmid/recombinant vector, they will not
have the antibiotic resistance gene and will die in the presence of antibiotic, allowing for selection
and identification of the plasmid-containing cells.

Screenable marker for recombinant molecules:

Allows screening of different phenotypes.
An example of a screenable marker for recombinant molecules are genes which express a colour
change in the presence of a substrate, thus allowing screening of the different phenotypes.

3) Unique restriction endonuclease (RE) sites (sometimes called restriction sites).

Allows inserts to be cloned in specific sites on the plasmid.

4) Promoters for gene expression.

Allows expression of a cloned gene in the vector.

- Vectors are self-replicating DNA molecules used to transfer foreign DNA between host cells.

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- Ideal vectors are small in size with at least one restriction endonuclease site or endonuclease
enzyme. Most cloning vector systems have been highly commercialised and have been used in
many research projects, and as such, the average vector has multiple endonuclease sites.
- Examples of vectors include:
1) Plasmids, which are contained in bacteria. They are extrachromosomal molecules that are
circular and double stranded. They are able to replicate independently of the genomic material in
the cell.
2) Bacteriophages, which are viruses that infect bacteria. Bacteriophages multiply within the host
cell and are released from the host cell as they multiply. If the bacteriophage contains a gene of
interest, it too will multiply with the bacteriophages.
3) Cosmids, which are specialised plasmids with DNA sequences called cos sites, to which foreign
DNA can be inserted.

The Development of Recombinant

Insulin
*cDNA is complementary DNA.

Applications of Recombinant
Proteins
Medical applications:
- Haematopoietic growth factor (to do with making blood).
- Hormones.
- Interferons (signalling proteins involved in the immune system).
- rProteins.
- Tissue/bone growth factors and clotting factors.
- Biological response modifiers.
- Medical research.

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Kinases and Phosphatases – Week 3 Lecture 3
Introduction to enzymes:
- Most enzymes are proteins.
- Some enzymes are RNA molecules and are called Ribozymes. An example of this is Peptidyl
Transferase which catalyses the formation of the protein bond in protein synthesis.
- Enzymes are highly specific catalysts that accelerate the rate of a reaction. They produce a new
product from the substrate that they act on. They enhance the specificity of that metabolic
chemical reaction.
- Kinases are an example of enzymes.
Enzymes
- Enzymes have an active site within the protein to which the substrates (target molecule) of the
reaction bind.
- In the case of kinases, the substrates are the target protein and ATP, and they would bind to the
target site of that enzyme.
- Enzymes reduce the amount of activation energy needed to reach a transition state, thereby
speeding up the reaction.
- Binding to the enzyme places strain on bonds in the substrate and lowers the energy needed to
break and form new bonds and hence form the products.
- In the case of kinases, the products would be a protein with a phosphate attached (Protein-P) and
ADP.
- For kinases:
Substrates Products
Target Protein + ATP ➔ Target Protein-P + ADP

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Kinases
- There are over 500 protein kinases in the human genome.
- Protein kinases comprise 2% of human genes.
- They have evolved from a common ancestral gene, and they share sequence similarity.
- Protein structural domains are important for their functions.
- 30% of human proteins can be modified by kinase activity.
- Kinases are enzymes that catalyse the phosphorylation (addition of phosphate) of proteins.
- Kinases take a phosphate from Adenosine Triphosphate (ATP) and link it to a protein, thereby
yielding a protein with a phosphate attached and Adenosine Diphosphate (ADP).
- This reaction is unidirectional because of the large amount of energy released when the phosphate
bond in ATP is broken.

- Kinases add the γ (gamma) phosphate from ATP (this is the

terminal phosphate) to an amino acid of the protein that has
an OH group in its variable side chain. This can be Serine,
Threonine or Tyrosine as these amino acids have OH groups in
their variable side chains.

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- Protein kinases phosphorylates a specific sequence motif. This means that is not just the Serine,
Threonine or Tyrosine that is recognised, but it is the sequence of amino acids on either side of the
target Serine, Threonine or Tyrosine that is recognised by the protein kinase.
- Each kinase recognises its own consensus sequence.
- Some kinases phosphorylate only one protein, whereas others can phosphorylate many proteins.
Thus, kinases activated by different signalling pathways can result in phosphorylation of the same
protein.

Protein Phosphorylation

- More than one site on a polypeptide chain can be phosphorylated. This

means there can be multiple amino acids within a protein that can be
phosphorylated.
- Phosphorylation adds two negative charges to a protein. Note the O - - →
- Recall that the structure of a protein depends on the interactions between
the variable side chains of its amino acids. Introducing a negative charge
onto that polypeptide chain will change how these different variable side chains interact with each
other. Other negatively charged amino acids in the vicinity will move away from these
phosphorylated amino acids and positively charged amino acids will be attracted.
- Phosphorylation causes a conformational change in the protein and changes its shape, and the
shape of a protein determines its function.
- Thus, adding the phosphate group to a protein could change its:
- Activity (increase or decrease).
- Interactions with other proteins, because if its shape changes and it is now able to interact with
another protein, we can get a signal transduction pathway because proteins can now interact with
other proteins with which they previously could not.
- Location in which they are found.
- Phosphorylation is very important in signalling pathways, regulating degradation of proteins, and
regulation of the cell cycle.

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Kinase Catalytic Domain
- Recall that a domain is a portion of a protein that can fold stably into a 3D shape, and it has a
particular function.
- The kinase catalytic domain is the domain of an enzyme (which is a protein) that contains the
catalytic/active site.
- The catalytic domain folds into a two lobed structure, like a bean or kidney.
- The upper lobe is the N-terminal lobe (or the amino end), and it is small. Within the N-terminal is an
ATP binding loop that binds ATP.
- The ATP binding loop has a Lysine somewhere in it.
- The lower lobe is the C-terminal lobe (or the carboxyl end), and it is large. The C-terminal has a
catalytic loop that binds the target protein.
- The catalytic loop has an Aspartic Acid somewhere in it.
- The cleft between the two lobes is the site of catalysis.
- An activation loop is found in the C-terminal loop, and this blocks the catalytic cleft (where the
target protein binds) until the activation loop is phosphorylated.
- The activation loop flips outward when in it is phosphorylated and thus the catalytic loop becomes
available to bind the target protein.
- Once ATP and protein are bound to the kinase catalytic domain, catalysis can occur in the cleft.

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- Shown alongside in green is the
activation loop before it is
phosphorylated. It is blocking the
catalytic site.
- Shown in yellow is the outward
movement of the activation loop
after it has been phosphorylated. It
exposes the catalytic site, and the
kinase is thus active. It can perform
its function of phosphorylating a
protein.

Types of Protein Kinases

- Kinases are usually found either as receptors (they have two jobs, to be a kinase and to be a
receptor), or they themselves are not receptors but they are associated with receptors in the
membrane, or if they are in the cytoplasm, they phosphorylate receptor proteins or membrane
receptors.
- They are found early on in signalling pathways.
- Recall that kinases add a phosphate to Serine, Threonine, or Tyrosine amino acids.
- Types of kinases include:

1) Serine/Threonine Protein Kinases

- These kinases attach a phosphate to Serine and Threonine.
- Cytoplasmic (found in cytoplasm) Serine/Threonine Kinases include MAPK, AKT, PKA, and PKC.
- Receptor Serine/Threonine Kinases are part of a receptor. The receptor is a protein that also has
intrinsic Serine/Threonine Kinase activity. It is both a kinase and a receptor. Examples of Receptor
Serine/Threonine Kinases include TGFßRI and TGFßRII (Transforming Growth Factor Beta Receptor
1 and 2). They will phosphorylate their target proteins on Serine and Threonine.

2) Tyrosine Kinases
These kinases attach a phosphate to Tyrosine.
Tyrosine Kinases can be found inserted in the plasma membrane of the cell and act as receptors.
These are called Receptor Tyrosine Kinases, and an example is EGFR (Epidermal Growth Factor
Receptor).
There are also Tyrosine Kinases found in the membrane, but it is a separate protein, and it is
associated with another membrane receptor. This means that it itself is not a receptor, but it is
associated with another receptor. An important example of this is JAK (Janus Kinase).
Cytoplasmic Tyrosine Kinases attach a phosphate to a tyrosine amino acid of a protein found in the
cytoplasm. An example of this is SRC.

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Revision:
- Domain – A portion of tertiary structure that folds stably into a 3D
shape and has its own function. The domain is found within the
same polypeptide chain. Alongside is the full tertiary structure of
a single protein, and within it are the regions that fold stably.
These are the domains, and 3 domains can be seen in the
single protein.

- Subunit – A separate polypeptide chain that gives a

protein quaternary structure. Alongside is a protein
formed by 4 subunits (separate polypeptide chains).

Regulation of Kinases
- Kinases may have a regulatory (inhibitory) domain. This is part of the same polypeptide chain that
contains the catalytic domain. Thus, this kinase would have at least 2 domains, a bean-shaped
catalytic domain as explained previously, and a regulatory (inhibitory) domain. Examples of kinases
that have a regulatory domain include Calcium Calmodulin Protein Kinase and SRC Tyrosine Kinase.

- Kinases may have a regulatory subunit. This is another polypeptide that binds to the kinase
polypeptide/protein and regulates it. It masks the active site when bound. An example of this is PKA
(Protein Kinase A).

- Kinases may need another protein to bind to the kinase. This activates the kinase. An example of a
kinase that is activated by the binding of another protein is CDK (Cyclin Dependent Kinase), which
requires the binding of Cyclin.

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Examples of Kinases
1) Serine/Threonine Kinases

- Calcium Calmodulin Dependent Kinase

- As shown above, the regulatory domain is interacting with the catalytic domain, and is inhibiting it.
This kinase is thus inactive.
- When Calcium binds to Calmodulin, Calcium Calmodulin is formed.
- When Calcium Calmodulin binds to the regulatory domain, it will no longer interact with the
catalytic domain, and thus the Calcium Calmodulin Dependent kinase is active, as shown below.

- Protein Kinase A (PKA)

- PKA is a tetrameric (has 4 subunits) kinase.
- Two subunits are regulatory subunits, and two are catalytic subunits.
- cAMP (cyclic Adenosine Monophosphate) is produced in a signalling pathway, and it binds to the
regulatory subunits and changes their shape, causing them to dissociate and move away from the
catalytic subunits.
- The catalytic subunits would now be active, and the kinase can phosphorylate its target protein.

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- Cyclin Dependent Kinase (CDK)
- This kinase plays an important role in regulation of the cell cycle.
- CDK is a small protein composed of little more than a catalytic domain.
- Cyclin Dependent Kinase (CDK) is activated by binding of a cyclin.

2) Tyrosine Kinases

- Receptor Tyrosine Kinases

- Receptor Tyrosine Kinases are found in the membrane and the protein is both a receptor, and a
Tyrosine Kinase.
- Inactive Receptor Tyrosine Kinases are found as monomers in the cell membrane.
- Each monomer has three domains:
1) An extracellular domain, to which a ligand (small molecule with transmits signals between cells)
will bind.
2) A transmembrane domain that passes through the membrane.
3) An intracellular domain that has a catalytic domain (shown in red).
- Below the catalytic domain is a tail of tyrosine amino acids, which are in a sequence motif (a
sequence motif is the tyrosine amino acid and the other amino acids around it, and this sequence is
recognised by the kinase).
- When the ligand binds, dimerization occurs,
which is the combining of two monomers to
form a dimer.
- Due to interaction between the monomers
and the ligand, we get a change in shape
(conformational shape), which as you should
recall, always means a change in function.
- ATP can now access the active site in the
catalytic domain and the Tyrosine kinase
becomes active.
- The Tyrosine Kinase now phosphorylates Tyrosine (in the tail below the
catalytic site). The left catalytic site (shown in red) phosphorylates a
Tyrosine on the left tail, and the right catalytic site phosphorylates a
Tyrosine on the right tail. We say the Tyrosine Kinase autophosphorylates
itself on the cytoplasmic tail Tyrosine amino acids.

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- SRC Tyrosine Kinase
- SRC Tyrosine Kinase is found in the cytoplasm.
- It has 3 domains, 1 catalytic domain, and 2 regulatory
domains – SH2 and SH3 (shown in yellow and green in
the image).
- The SH2 domain can be seen interacting with the tail of
the protein.
- The SH3 domain can be seen interacting with and
inhibiting the catalytic domain.
- The two inhibitory/regulatory domains are interacting
with and inhibiting the catalytic domain, and thus the
SRC Tyrosine Kinase is inactive.

- To activate the SRC Tyrosine Kinase:

The phosphate on the tail which was interacting with
the SH2 domain must be removed.
A protein (shown in orange) comes and binds to the SH2
domain and moves it away from the catalytic domain.
Phosphorylation of the activation loop must occur, and
the activation loop will flip outward from the catalytic
cleft.
- The SRC Tyrosine Kinase is now active and can
Phosphorylate its target Tyrosine.

Structure
- You do not need to know their full structure – simply see the side chains (attached to central C).
- Note that three amino acids that can be phosphorylated by kinases all contain OH.

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Phosphoprotein Phosphatases
- These are enzymes that dephosphorylate (remove phosphate) proteins that have been
phosphorylated.
- They catalyse dephosphorylation.

- Unlike kinases that developed from a common ancestor, phosphatases have evolved from separate
families. Phosphatases are completely separate enzymes.
- Similarly to how we have 2 types of kinases (Serine/Threonine Kinases and Tyrosine Kinases), we
have 2 phosphatases:

1) Phosphoserine/Phosphothreonine Phosphatases
- These are heteromeric (subunits are different to each other) proteins consisting of a catalytic
subunit associated with regulatory subunits (they consist of more than one subunit).
- An example is PP2A which regulates cell proliferation by removing the phosphate from proteins in
signalling pathways that have been activated by growth factors.
- Another example is PP2B (Calcineurin) which has a regulatory subunit that binds Calcium (Ca 2+)

2) Phosphotyrosine Phosphatases
- There are about 100 protein Tyrosine Phosphatases.
- They have very specific substrates.
- There are two main classes of Phosphotyrosine Phosphatases:
1) Intracellular (such as PTP1B – Phosphotyrosine Phosphatase 1 B).
2) Receptor-linked which are proteins within the membrane (such as CD45 Leucocyte Common
Antigen).

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Cellular Communication – Week 3 Lecture 4
Intercellular Communication
- Intercellular communication is communication between cells.
- The billions of cells in our body requires communication so that they receive the right information
at the right time so that they can work together.
- Without proper communication, cells will not function correctly.
- We will look at 5 types of intercellular communication:
1) Autocrine Signalling – Same cell
- A cell secretes signalling molecules, and these signalling molecules
bind to cell surface receptors on the same cell that produced them.

2) Paracrine signalling – Local cells

- A cell releases signalling molecules into the extracellular space, and
these signalling molecules act locally on neighbouring cells.
- These signalling molecules are rapidly degraded so that the signal
does not travel further away.

3) Endocrine signalling – Far away cells

- Endocrine cells secrete hormones into the bloodstream that are
then distributed widely throughout the body.
- The target cell is usually far from the endocrine cell.

4) Contact-dependent signalling/ Cell to Cell signalling

- This is signalling between 2 (usually epithelial) cells.
- There is cell membrane to cell membrane contact, as cells need to be
in direct contact with each other.

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5) Synaptic signalling
- This is signalling performed by neurons that transmit signals
electrically along their axons and release neurotransmitters at
synapses, which are often located far away from the cell body.

Summary

Why do cells need to communicate?

- Cells need signalling for normal function. An example is for movement – your muscles have to co-
ordinate their contractions.
- Cells need signalling to tell them when to grow and divide (proliferation). This is required in the
developing foetus as well as in wound healing.
- Cells need signalling for differentiation (to become specialised). An example is stem cells in the
bone marrow that differentiate into blood cells.
- Cells need to be signalled when to die – to commit suicide. If a cell is a threat to the organism such
as cancerous cells or cells with damaged DNA, it must die so that it cannot harm the cell.
Programmed cell death is called apoptosis.

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Principles and Components of Hydrophobic Signal Transduction
Intracellular Receptor (Hydrophobic Signalling)
- This is signalling where the signalling molecule is hydrophobic (water-hating).
- If it is hydrophobic, it must mean it is a lipid, and these are usually steroid hormones. They can also
be thyroid hormones (contain tyrosine) and some vitamins.
- Hydrophobic signalling consists of 3 components:
- Ligands (the extracellular signalling molecule).
- Receptors.
- Transcription factors.
- Recall that the membrane of a cell is made of lipids, and so too is the steroid ligand. The steroid
ligand can thus diffuse through the membrane. The steroid ligand enters the cell and binds to a
specific intracellular receptor inside the cell.
- The ligand and receptor together form a transcription factor-like protein called the receptor-
hormone complex, which moves into the nucleus, binds to ta hormone-response element
(resembles enhancer elements), and results in the production of specific proteins.
- Hydrophobic signalling results in the production of new signalling because we have initiated
transcription and translation.
- Learn the following:

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Principles and Components of Hydrophilic Signal Transduction
Membrane Receptor (Hydrophilic signalling)
- This is signalling where the signalling molecules are hydrophilic (water-loving).
- These molecules are soluble in fluid, and thus cannot pass through our lipid membrane.
- Hydrophilic signalling molecules are usually protein or polypeptide based.
- Thus, the protein/polypeptide ligands bind to specific receptors present on the cell membrane.
- That transmembrane receptor converts the extracellular signal into an intracellular chain of
biochemical events which amplifies the signal.
- For this signalling pathway, information is conveyed through the cytoplasm through one of two
ways:
- Through diffusible elements (such as Calcium of cAMP) called second messengers.
- Through protein-protein interactions via their domains resulting in signal transduction.
- Hydrophilic signalling pathways result in the activation of a tyrosine kinase.
- The final resulting outcome of signalling through a hydrophilic signalling pathway is proteins with
altered function, or transcription factors are activated, resulting in the production of new proteins.
- In hydrophobic signalling, new proteins are produced, not proteins with altered function.
Interaction Domains in Signal Transduction
- Different proteins interact through their domains. The domains of one protein interact with the
domains of another.
- There are approximately 40 to 50 different domains. We will focus on a few:
- DNAB – DNA Binding domain. These Domains bind to DNA and an example of where a DNA
Binding domain can be found is in transcription factors.
- Catalytic Domain – We have covered this already.
- PH – Plecktrin homology domain – This domain binds to PI-3,4,5-trisP (Phosphoinositol-3,4,5-
trisPhosphate), which is a phospholipid present in the cell membrane.
- SH2 – SRC homology 2 domain – SH2 binds to a phosphorylated Tyrosine. Note that it is not just
the amino acid Tyrosine, it is the amino acids around the Tyrosine called the peptide motif that it
binds to. The SH2 binds to a peptide motif containing a phosphorylated Tyrosine.

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Hydrophilic signalling continued:
- An extracellular signalling molecule called a ligand (usually a protein or polypeptide) binds to the
membrane receptor.
- The receptor transduces the signal into the cell where further signalling will occur.
- An adaptor protein links the membrane receptor to other proteins in the cell cytoplasm, such as a
monomeric G protein.
- Heterotrimeric G proteins link directly to the membrane receptor.
- The Heterotrimeric G protein results in the production of a second messenger.
- The second messenger or the monomeric G protein can activate a kinase.
- The kinase can then phosphorylate another protein and alter its function, or it can phosphorylate a
transcription factor to activate it that activated transcription factor will move into the nucleus and
bind to the promoter of genes. We will then get transcription of those genes and a new protein will
be made.
- Scaffold proteins can also be found in signalling pathways, and they bind numerous proteins
together and increase the efficiency of signalling in that signalling pathway.

Components of Hydrophilic Signalling

1) Ligands
- Ligands are extracellular signalling molecules that bind to specific receptors.
- They are known as 1st messengers or hormones.
- Examples include chemokines (signalling molecules that cause movement of cells), cytokines
(signalling molecules used between cells of the immune system), growth factors (signalling
molecules that cause proliferation of cells), and hormones (signalling molecules that results
different physiological function effects related to the specific hormone).
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2) Membrane Receptors
- If a receptor is not present, the ligand will not bind to it, and we will not get a signal.
- The response depends on the relative numbers of receptors for that hormone or in the target cells,
and the affinity (strength) of the bond between the hormone and the receptor. A greater afiinity
will result in a greater signal.
- Hydrophilic ligand receptors have extracellular domains that interact with the specific ligand (recall
that this is because the ligand is hydrophilic and cannot pass through the membrane, and so the
receptor must have a piece of itself outside of the cell).
- Hydrophilic ligand receptors have domains/motifs (a sequence of amino acids) in the cytoplasm
that can transduce the signal from outside to inside because the domain/motif interacts with
intracellular signalling molecules.
- There are three main types of membrane receptors:
1) Those without kinase activity, such as GPCR (G Protein Coupled Receptors).
2) Those with intrinsic kinase activity (the receptor protein itself is a kinase), such as Receptor
Tyrosine Kinase.
3) Those associated with a kinase (this receptor is bound to a separate protein that is a kinase),
such as Cytokine receptors, T Cell Receptors, and B Cell Receptors.

3) Adaptors
- Adaptors are relatively small proteins with usually no more than two or three domains whose
function is to link two proteins together.
- Quite often, adaptor proteins have an SH2 domain (recall that SH2 domains bind to phosphorylated
Tyrosine amino acids). Different SH2 domains recognise the P-Tyrosine (phosphorylated Tyrosine)
associated with different amino acids forming the sequence motifs.
- Shown alongside is an adaptor protein with 3 domains. The SH2
domain will bind to a P-Tyrosine and the amino acids around it,
and one of the other domains will bind to the next protein in
that signalling pathway.

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- Adaptor functioning
- A receptor is phosphorylated on a Tyrosine amino acid.
- An adaptor protein with an SH2 domain comes along and binds to the P-Tyrosine and the amino
acids surrounding it.
- Other domains of the adaptor protein interact with domains of the next protein in that signalling
pathway.

4) G Protein
- G proteins get their name from the fact that they bind GTP (Guanosine Trisphosphate) or GDP
(Guanosine Diphosphate). If it bound to ATP, it would be called an A protein.
- When proteins are bound to GDP, they are inactive.
- G proteins are activated by the exchange of GDP for GTP (G proteins are active when GTP is bound).
Note that GDP does not get changed into a GTP. The entire GDP is replaced by a GTP.
- If G proteins are active, they can pass the signal on.
- G proteins have intrinsic GTPase activity. This means that they can convert GTP back to GDP by
removing the terminal phosphate. In doing so, they switch themselves off. They are no longer
active, and the signalling pathway is stopped.
- There are two types of G proteins:

1) Small Monomeric (one subunit) such as

RAS.
- Signalling through hydrophilic pathways
recruits a GEF (Guanine Exchange Factor)
which exchanges GDP for GTP on the
monomeric G protein RAS. GDP is released.
This activates RAS.
- RAS’s intrinsic GTPase activity converts GTP
back to GDP by removing the terminal
phosphate of GTP. RAS is now bound to GDP
and no longer active.

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2) Heterotrimeric (three different subunits) G Protein
- Consists of an α subunit, a ß subunit, and a γ subunit.
- The Alpha (α) subunit binds GTP/GDP.
- If GDP is bound, the heterotrimeric G protein is inactive.
- When a ligand (such as a hormone) binds to the receptor (called a G Protein coupled receptor,
because heterotrimeric G proteins link directly to the receptor) changes shape and the GDP is
released and a GTP is attached to the α subunit. The G protein is now
active.
- The trimer dissociates into an α subunit with the GTP attached, and
the ß and γ subunit.
- Recall that G proteins have intrinsic GTPase activity. The α subunit can
convert the GTP back to GDP by removing the terminal/gamma
phosphate from the GTP.
- This produces an α subunit with GDP that is inactive.
- The ß and γ reassociate with the α subunit, and we are back to
having our inactive heterotrimeric G protein.

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5) Second Messengers
- Activation of heterotrimeric G proteins (with GTP attached) results in activation of effector
enzymes.
- Effector enzymes (proteins that catalyse reactions and produce products) produce second
messengers, which are diffusible chemicals such as cAMP and IP3 (Inositol Trisphosphate).
- These second messengers then activate kinases.
6) Protein Kinases
- Recall that kinases are enzymes that phosphorylate proteins.
- Kinases transfer the terminal phosphate from ATP to a Serine, Threonine, or Tyrosine amino acid on
the protein.
- This reaction is unidirectional (only in one direction) because of the large amount of energy
released when the phosphate bond in ATP is broken.
- The kinase adds two negative charges to the protein. This changes its shape and in turn its activity,
location, or interactions with other proteins.

7) Scaffold Proteins
- Scaffold proteins are large proteins that can bind various other proteins to bring them together in a
complex.
- Scaffold proteins have numerous domains that can bond many other proteins to them.
- Scaffold proteins have no enzymatic activity.
- They can be phosphorylated at various sites and thus recruit many different proteins with SH2
domains (recall that SH2 domains only bind to Tyrosine amino acids that have been
phosphorylated).
- Because we have several proteins now bound together, we can increase the efficiency of signalling
through signalling pathways.
8) Transcription Factors
- Recall that transcription factors are proteins that bind to promoters of genes and either increase or
decrease transcription of a gene.
- The promoter contains several conserved consensus sequences (such as TATA and CAAT) which
bind general transcription factors, as well as other specific transcription factors to specific
sequences within those promoters.
- Specific transcription factors give specificity to the signal. They determine whether those genes are
expressed or not.
- Genes with promoters with the same sequences will bind the same transcription factors, giving a
combined response.
- We need specific transcription factors as well as general transcription factors bound to initiate
transcription of a gene.

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Speed of Signalling Pathways
- Hydrophobic signalling has far less components that Hydrophilic signalling.
- Which is faster depends on what is happening.
- Recall that hydrophobic signalling only produces new proteins, whereas hydrophilic signalling can
alter proteins, or produce new proteins.
- If a protein is being altered, it will be a very quick process, as the protein already exists.
- If a new protein is being produced, it will be a lengthy process as transcription and translation take
time.

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