REGULATION OF GENE

BIO 171 LEC EXPRESSION

Esmeralda, Lovely A.
(Cell and Molecular Biology
Lecture)

Gene TRANSPOSABLE ELEMENTS

Mandawe, Vea T.

Expression SPLIT GENES AND RNA

Esmeralda, Lovely A. SPLICING
Jalil, Alwira Hannah Sheridan S.
Flores, Janus C.
Jalil, Alwira Hannah Sheridan S.
Mandawe, Vea T.
ORIENTATION AND
Mohammad Ali, Raghad A.
GROUP 3 – BS BIOLOGY 3A RECOGNITION OF TATA BOX
Mohammad Ali, Raghad A.

TRANSCRIPTIONAL
REPRESSION, RNA STABILITY,
AND PROTEIN ACTIVITY
Flores, Janus C.
REGULATION OF GENE
EXPRESSION
ESMERALDA, LOVELY A.
 REGULATION OF GENE EXPRESSION

• THE PROCESS OF CONTROLLING WHICH GENES IN A CELL’S DNA ARE EXPRESSED IS KNOWN
AS GENE REGULATION (USED TO MAKE A FUNCTIONAL PRODUCT SUCH AS A PROTEIN).
• GENE EXPRESSION IS THE PROCESS OF TURNING ON A GENE TO PRODUCE RNA AND
PROTEIN.
• EVEN THOUGH ALL CELLS IN A MULTICELLULAR ORGANISM HAVE THE SAME DNA, THEY MAY
EXPRESS SUBSTANTIALLY DIVERSE SETS OF GENES.
• A CELL’S SET OF EXPRESSED GENES DEFINES THE PROTEINS AND FUNCTIONAL RNAS IT
CARRIES, GIVING IT ITS DISTINCT CHARACTERISTICS.
 GENE REGULATION
• GENE REGULATION IS THE PROCESS BY WHICH A CELL DETERMINES WHICH OF THE
NUMEROUS GENES IN ITS GENOME ARE "TURNED ON" (EXPRESSED).
Ex. Liver cell and Neuron
PROKARYOTIC VS. EUKARYOTIC GENE REGULATION
 IN PROKARYOTIC CELLS...
• PROKARYOTIC GENE EXPRESSION IS PRIMARILY CONTROLLED AT THE LEVEL OF TRANSCRIPTION

• PROKARYOTIC GENE EXPRESSION (BOTH TRANSCRIPTION AND TRANSLATION) OCCURS WITHIN THE
CYTOPLASM OF A CELL DUE TO THE LACK OF A DEFINED NUCLEUS; THUS, THE DNA IS FREELY
LOCATED WITHIN THE CYTOPLASM.
• TO SYNTHESIZE A PROTEIN, THE PROCESSES OF TRANSCRIPTION (DNA TO RNA) AND TRANSLATION
(RNA TO PROTEIN) OCCUR ALMOST SIMULTANEOUSLY. WHEN THE RESULTING PROTEIN IS NO
LONGER NEEDED, TRANSCRIPTION STOPS. THUS, THE REGULATION OF TRANSCRIPTION IS THE
PRIMARY METHOD TO CONTROL WHAT TYPE OF PROTEIN AND HOW MUCH OF EACH PROTEIN IS
EXPRESSED IN A PROKARYOTIC CELL.
 IN EUKARYOTIC CELLS...
• EUKARYOTIC GENE EXPRESSION IS CONTROLLED AT THE LEVELS OF EPIGENETICS, TRANSCRIPTION, POST-
TRANSCRIPTION, TRANSLATION, AND POST-TRANSLATION

• EUKARYOTIC GENE EXPRESSION OCCURS IN BOTH THE NUCLEUS (TRANSCRIPTION) AND CYTOPLASM (TRANSLATION).

• DNA IS CONTAINED INSIDE THE CELL’S NUCLEUS WHERE IT IS TRANSCRIBED INTO RNA. THE NEWLY-SYNTHESIZED RNA
IS THEN TRANSPORTED OUT OF THE NUCLEUS INTO THE CYTOPLASM WHERE RIBOSOMES TRANSLATE THE RNA INTO
PROTEIN. THE NUCLEAR MEMBRANE PHYSICALLY SEPARATES THE TRANSCRIPTION AND TRANSLATION PROCESSES;
TRANSCRIPTION TAKES PLACE ONLY WITHIN THE NUCLEUS, WHEREAS TRANSLATION TAKES PLACE ONLY OUTSIDE THE
NUCLEUS IN THE CYTOPLASM. GENE EXPRESSION CAN BE REGULATED AT ANY POINT DURING THE PROCESS. WHEN
THE DNA IS UNCOILED AND LOOSENED FROM NUCLEOSOMES TO ALLOW TRANSCRIPTION FACTORS TO BIND
(EPIGENETICS), WHEN THE RNA IS TRANSCRIBED (TRANSCRIPTIONAL LEVEL), WHEN THE RNA IS PROCESSED AND
EXPORTED TO THE CYTOPLASM AFTER IT IS TRANSCRIBED (POST-TRANSCRIPTIONAL LEVEL), WHEN THE RNA IS
TRANSLATED INTO PROTEIN (TRANSLATIONAL LEVEL), OR AFTER THE PROTEIN HAS BEEN MADE (POST-TRANSLATIONAL
LEVEL), WHEN THE RNA IS TRANSLATED INTO PROTEIN (TRANSLATIONAL (POST-TRANSLATIONAL LEVEL).
 PROKARYOTIC GENE REGULATION
• MOST PROKARYOTIC GENES ARE REGULATED IN UNITS CALLED OPERONS.
• AN OPERON IS COMPOSED OF A PROMOTER, AN OPERATOR, AND THE STRUCTURAL GENES.
• REPRESSORS ARE PROTEINS THAT SUPPRESS TRANSCRIPTION OF A GENE IN RESPONSE TO AN EXTERNAL
STIMULUS. IN OTHER WORDS, A REPRESSOR KEEPS A GENE “OFF”
• ACTIVATORS ARE PROTEINS THAT INCREASE THE TRANSCRIPTION OF A GENE IN RESPONSE TO AN EXTERNAL
STIMULUS. IN OTHER WORDS, AN ACTIVATOR TURNS A GENE “ON”
• INDUCERS ARE SMALL MOLECULES THAT EITHER ACTIVATE OR REPRESS TRANSCRIPTION DEPENDING ON THE
NEEDS OF THE CELL AND THE AVAILABILITY OF SUBSTRATE. INDUCERS BASICALLY HELP SPEED UP OR SLOW
DOWN “ON” OR “OFF” BY BINDING TO A REPRESSOR OR ACTIVATOR. IN OTHER WORDS: THEY DON’T
WORK ALONE.
THE TRP OPERON: A REPRESSOR OPERON
CATABOLITE ACTIVATOR PROTEIN (CAP): AN
ACTIVATOR REGULATOR
 ACTIVATORS AND REPRESSORS

• ACTIVATORS (AND SOMETIMES INDUCERS) INSTIGATE POSITIVE REGULATION

• REPRESSORS INSTIGATE NEGATIVE REGULATION
• ACTIVATOR OR INDUCER ENABLE OR INCREASE THE RATE TRANSCRIPTION OF A CERTAIN
PROTEIN
• REPRESSOR SLOW OR STOP THE TRANSCRIPTION
EUKARYOTIC GENE REGULATION

LEVELS OF GENE REGULATION IN EUKARYOTES

EPIGENETIC LEVEL
TRANSCRIPTIONAL LEVEL
POST-TRANSCRIPTIONAL LEVEL
TRANSLATIONAL LEVEL
POST-TRANSLATIONAL LEVELS.
 Mandawe, Vea T.
BS BIO 3A
WMSU
What are transposable elements?
• AKA "jumping genes" or transposons
• Sequences of DNA that move or jump from one location in the
genome to another.
Two Major Classes
Class 1: Retrotransposons
Class 2: DNA transposons

14
Class 1: Retrotransposons
Follow ‘copy-and-paste’ mechanism
Copied in 2 stages
• First, they are transcribed from DNA to RNA, and the RNA
produced is then reverse transcribed to DNA.
• into the genome at a new position. The copied DNA is then
inserted back
• The reverse transcription step is catalyzed by a reverse
transcriptase, which is often encoded by the TE itself.
• The characteristics of retrotransposons are similar to
retroviruses, such as HIV.

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Class 1: Retrotransposons
Grouped into three main orders:
• Long Terminal Repeat (LTR) retrotransposons- encode reverse transcriptase,
similar to retroviruses
Non-LTR retrotransposons
• Long Interspersed Nuclear Elements (LINEs)- encode reverse transcriptase but
lack LTRs, and are transcribed by RNA polymerase II
• Short Interspersed Nuclear Elements (SINEs)- do not encode reverse
transcriptase and are transcribed by RNA polymerase III.

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Class 2: DNA transposons
• First TE discovered by Barbara McClintock in maize
• Use a cut-and-paste transposition mechanism
• Does not involve an RNA intermediate
• The transposition are catalyzed by several transposase enzymes
Mechanism of transposition
• The transposase makes a staggered cut at the target site producing sticky ends,
• Transposases cuts out the DNA transposon and ligates it into the target site.
• A DNA polymerase fills in the resulting gaps from the sticky ends and
• DNA ligase closes the sugar-phosphate backbone.
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Autonomous and Nonautonomous Transposons
• Autonomous TEs- Do not require other elements for
their mobility. Hence, they can move on their own.
Example: Ac elements
• Nonautonomous TEs- require the presence of other TEs
in order to move from locus to another.
Example: Ds elements
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Importance of Transposable Elements
• A transposon or a retrotransposon that inserts itself into a
functional gene can disable that gene.
• After a DNA transposon leaves a gene, the resulting gap may not
be repaired correctly.
• Multiple copies of the same sequence, such as Alu sequences can
hinder precise chromosomal pairing.
• Many TEs contain promoters which can cause aberrant expression
of linked genes, causing disease or mutant phenotypes.
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TEs are mutagens and their movements often cause
genetic diseases.
• Hemophilia A and B
• Severe combined immunodeficiency
• Porphyria
• Predisposition to cancer
• Duchenne muscular dystrophy
• Alzheimer’s disease
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Applications of TEs
• Used for analysis of gene expression and protein functioning in signature-tagging
mutagenesis.
• TEs can be used in vectors to insert a sequence in novel position
• Just like spotted seed of maize, TEs are also used to understand the role of same gene
in different cells of an organism
• The Sleeping Beauty transposon system has been used extensively as an insertional
tag for identifying cancer genes
• Potentially used in human gene therapy.
• Used for he reconstruction of phylogenies by the means of presence/absence
analyses.

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Split Genes
and RNA
Splicing
Alwira Hannah Sheridan S. Jalil
Group 3 - BS Biology 3A
Discovery of Split Genes
• discovered by Richard J. Roberts and Phillip A. Sharp in 1977
• studied hybrid of adenovirus mRNA and one (template)
strand of DNA under the electron microscope
• mRNA was found to hybridize with discontinuous stretches
Richard J. Roberts
of genomic DNA, and these intervening DNA stretches were
in loops, representing introns.
• implied the existence of then-unknown machinery
for splicing out introns and assembling genes (spliceosome)
• 94% of human genes are interrupted
• 50% of hereditary diseases are involved in splicing intron
errors out of interrupted genes (e.g. Beta-thalassemia)
Phillip A. Sharp
 Split Genes / Interrupted Genes
• genes that contain exons (coding sequences) that are
interrupted by introns (non-coding sequences)
• discontinuous and longer than mature RNA product due
to presence of introns
• present in eukaryotes, rarely in prokaryotes; making
eukaryotic genomes largely complex and inconsistent
• structure allows for the process of splicing, where various
mRNA products can be produced from a single gene
 INTRONS (Intervening Regions)
Non-coding DNA sequences present within a gene that are removed by
the process of RNA splicing during maturation of the RNA transcript.
❖ Denote both the DNA sequences within the gene and the corresponding
sequence in RNA transcripts.
❖ Common in the protein-coding nuclear genes of most jawed invertebrates
and other eukaryotic organisms, along with unicellular organisms like
bacteria.
❖ Introns have a donor site (5′ end), a branch site (near the 3′ end), and an
acceptor site (3′ end) that are required for splicing.
❖ During RNA splicing, the introns between the exons are removed to connect
two different exons that then code for messenger RNA.
❖ Introns are crucial because the variation in the protein bio-product formed is
greatly enhanced by alternative splicing in which introns take part in
prominent roles.
INTRONS (Intervening Regions)
 EXONS (Expressed Regions)
Protein-coding DNA sequences that contain the necessary codons or
genetic information essential for protein synthesis.
❖ Represents the expressed region present in the genome.
❖ Exosome – entire set of all exons present in the genome of the organisms.
❖ In genes coding for proteins, exons include both the protein-coding sequence
and the 5’ and 3’ untranslated regions.
❖ Found in all organisms ranging from jawed vertebrates to yeasts, bacteria,
and even viruses.
❖ In the human genome, exons account for only 1% of the total genome while
the rest is occupied by intergenic DNA and introns
❖ Alternative splicing enables exons to be arranged in different combinations,
where different configuration results in different proteins.
❖ Exon shuffling – exons or sister chromosomes are exchanged during
recombination.
 SPLICEOSOMES
Large and complex molecule formed of RNAs and proteins that regulate
the process of RNA splicing.
❖ Composed of five small nuclear RNAs (snRNA) and about 80 protein molecules.
❖ Combination of RNAs with these proteins results in the formation of an RNA-protein
complex termed as small nuclear ribonucleoproteins (snRNPs).
❖ Mostly confined within the nucleus where they remain associated with the immature
pre-mRNA transcripts.
❖ The spliceosome functions as an editor that selectively cuts out unnecessary and
incorrect materials (introns) to produce a functional final-cut.
❖ All spliceosomes are involved in both the removal of introns and the ligation of
remaining exons.
❖ These spliceosomes, in addition to working on RNA-RNA interactions, are also involved
in RNA-protein interactions.
SPLICEOSOMES
 RNA Splicing
Form of RNA processing in which a newly made precursor messenger
RNA (pre-mRNA) is transformed into a mature RNA (mRNA) by removing
the non-coding sequences termed introns.
• Involves the removal of non-coding sequences or introns and joining of the
coding sequences or exons.
• Takes place during or immediately after transcription within the nucleus in the
case of nucleus-encoded genes.
• In eukaryotic cells, RNA splicing is crucial as it ensures that an immature RNA
molecule is converted into a mature mRNA molecule that can then be
translated into proteins. The post-transcriptional modification is not necessary
for prokaryotic cells.
• Controlled process that is regulated by various ribonucleoproteins
RNA Splicing
Mechanism
 RNA Splicing Mechanism
• Introns possess a highly conserved GU sequence at their 5’
end, known as the donor site, and a highly conserved AG
sequence at the 3’ end, called the acceptor site.
• A large RNA-protein complex, the spliceosome, made up of
five small nuclear ribonucleoproteins (snRNPs) recognize
the start and end points of the intron thanks to these sites,
and catalyze the removal of the intron accordingly.
• The spliceosome forms the intron into a loop that can be
cleaved easily, and the remaining RNA on each side of the
intron is connected.
RNA Splicing Mechanism
RNA Splicing Mechanism
RNA Splicing Mechanism
RNA Splicing Mechanism
RNA Splicing Mechanism
RNA Splicing Mechanism
RNA Splicing Mechanism
 RNA Splicing Mechanism
1. U1 snRNP recognize and break the 5’ splice site of the
intron and bring it closer to branch site.
2. Complex of snRNP of U2, U4, U5 and U6 bind to the intron.
The complex of snRNPs and precursor mRNA of the intron
is called spliceosome.
3. The spliceosome is looped out. This loop of intron is called
lariat which is discarded and degraded.
4. The exons on either side of the removed intron are
brought closer and ligation seals them together.
 TYPES OF RNA SPLICING

Self- Exon
Splicing Skipping

Alternative Intron
Splicing Retention
Self-Splicing
➢ In some cases, specially group I
introns, the intron itself folds and
catalyzes its own splicing.
➢ RNA of intron functions as an
enzyme (ribozyme) and behaves
like an endonuclease to splice out
the intron.
➢ Splicing of intron sequences
requires no other enzyme.
➢ Intron is released in a linear form
which is subsequently degraded.
Alternative Splicing
➢ Different combinations
of exons are joined
together during the final
stages of transcription so
that more than
one messenger RNA is
produced from a single
gene.
➢ Allows a single gene to
code for multiple
proteins.
Exon Skipping
➢ Form of RNA splicing used
to cause cells to “skip”
over faulty or misaligned
sections of genetic code,
leading to a truncated but
still functional protein
despite the genetic
mutation.
Intron Retention
➢ Introns, rather than being spliced out as usual, are
retained in mature mRNAs.
➢ Helps in increasing gene product diversity and
regulating transcript functionality.
RNA Splicing Errors
• The splicing of nuclear pre-mRNAs is a fundamental
process required for the expression of most metazoan
genes. However, errors in splicing might occur due to
mutations that result in various splicing-related diseases.
• Mostly in alternative splicing, an erroneous splicing result
in biological products that are not functional.
• Errors during splicing might occur due to mutations at the
splice site, which causes loss of exons or inclusion of an
intron disrupting the function of the RNA sequence.
• Similarly, displacement of a splice site might also cause
the formation of longer or shorter exons, resulting in
erroneous products.
RNA Splicing Errors
• In living organisms like plants, stress-induced alternative
splicing associated with various metabolic pathways
might bring changes in the normal functioning of the
plant.
• The chances of erroneous splicing is more in eukaryotic
cells with high levels of alternative splicing containing
splice sites that have evolved to offer a weak binding
potential for components of the spliceosome
• The accuracy of splice-site pairing is also limited by the
accuracy of transcription as the transcription machinery
makes a mistake once in every 103–105 nucleotide
insertion step.
 RNA Splicing Applications
Pre-mRNA splicing is a fundamental process in cellular metabolism that plays an
essential role in generating protein diversity. The diversity is brought about by
changes in the number and sequence of exons and introns present in the RNA
sequence.
• RNA splicing also helps in the regulation of gene and protein content in the
cell.
• Splicing of RNA sequences assists the process of evolution of new and
improved proteins.
• Various aberrant splicing isoforms act as markers for cancer and as targets for
cancer therapy.
• Pre-mRNA splicing is a key to the pathology of cancers where it regulates the
three functional aspects of cancer: proliferation, metastasis, and apoptosis.
TATA Box
Orientation and
Recognition
MOHAMMAD ALI, RAGHAD A.
What is the TATA Box
 The TATA box is a sequence (5'-TATAAA-3') of DNA found in the core
promoter region of genes of archaea and eukaryotes.
 In bacteria, there is also an equivalent to the TATA Box of
eukaryotes and it is called the Pribnow- Schaller box or simply the
Pribnow Box.
 The segment is six- eight base pairs long and the nucleotides most
commonly found are TATAAAA
 The TATA box got its name as it is composed of sequence
characterized by repeating T (Thymine) and A (Adenine) base pairs.
Discovery of the TATA Box
 Another term for TATA Box is the Goldberg-Hogness box,
named after the people that first discovered it.
 The TATA box was the first eukaryotic core promoter identified
in 1978 by American biochemist David Hogness and his
graduate student, Michael Goldberg at the University of Basel
in Switzerland. They first discovered the TATA sequence while
analyzing 5’ DNA promoter sequences in Drosophilia
mammalian, and viral genes.
Location of the TATA Box

 The TATA box is usually located 25-35 base pairs upstream

(5' to the coding region) of the transcription start site.
 The TATA-box is the site of preinitiation complex formation, which is
the first step in transcription initiation in eukaryotes.
 The TATA box, as a core promoter element, is the binding site for a
transcription factor known as TATA-binding protein (TBP), which is
itself a subunit of another transcription factor: Transcription Factor II
D (TFIID).
 After TFIID binds to the TATA box via the TBP, five more transcription
factors and RNA polymerase combine around the TATA box in a
series of stages to form a pre-initiation complex.
Janus Flores

Transcriptional Repression,
RNA stability, and Protein
activity
 Transcriptional repression

Transcriptional repression is an essential mechanism in

the precise control of gene expression. Nearly 40 years
ago, Jacob and Monod recognized the importance of
transcriptional repressor molecules in the regulation of
gene expression in bacteria. While these initial studies
focused on regulation of the lactose operon
of Escherichia coli, it was soon realized that
transcriptional repression is a general mechanism
affecting gene expression in prokaryotes. Because the
basic mechanism of transcription in bacteria and
eukaryotes is remarkably well conserved, it came as no
surprise that gene-specific repressors were later
identified in eukaryotes
 Eukaryotic repressors, like activators, are
typically modular, consisting of either a
single polypeptide with functionally
distinct domains or multisubunit
complexes with distinct functions
distributed among the subunits. These
domains (or polypeptides) can target
different components of the transcription
machinery to affect distinct steps in
initiation (as shown in figure 1).
Thus, multiple mechanisms built within a single repressor ensure that
a gene can be silenced in an efficient manner. In this review we
concentrate on repressors that affect the formation of the initiation
complex. Repressors that target the transcriptional machinery
subsequent to initiation have also been described, but are beyond the
scope of this review.
 RNA Stability
The structural variations between RNA and DNA underlie the differences in
their stability and longevity. Because DNA is double-stranded, it is
inherently more stable. The single-stranded structure of RNA is less stable
but also more flexible and can form weak internal bonds. Additionally,
most RNAs in the cell are relatively short, while DNA can be up to 250
million nucleotides long. RNA has a hydroxyl group on the second carbon
of the ribose sugar, increasing the likelihood of breakage of the sugar-
phosphate backbone.

Recently, scientists have discovered an important role for methylation in mRNA

stability. The methylation of adenosine residues (m6A) appears to increase
mRNA translation and degradation. m6A also has roles in stress responses,
nuclear export, and mRNA maturation.
Protein Activity

The degradation of
proteins is
mediated by a
special class of
enzymes (proteins)
known as
proteases.
 S E E Y O U O N N E X T P R E S E N TAT I O N

T H A NK YO U
THANK YOU FOR LISTENING!