Transcription

Cell

Transcription
DNA
mRNA
-It is the process of creating a complementary RNA copy of a sequence
of DNA, because both RNA and DNA are nucleic acids, which use base
pairs of nucleotides as a complementary language that can be converted
back and forth from DNA to RNA by the action of the correct enzymes.
-During transcription, a DNA sequence is read by RNA polymerase,
which produces a complementary, antiparallel RNA strand.
-As opposed to DNA replication, transcription results in an RNA
complement that includes uracil (U) in all instances where thymine (T)
would have occurred in a DNA complement.
- Transcription can be explained easily in 5 simple steps:
1- DNA unwinds/"unzips" as the hydrogen bonds break.
2-The nucleotides of the RNA, pair with complementary DNA bases.
3- RNA sugar-phosphate backbone forms.
4- Hydrogen bonds of the untwisted RNA+DNA "ladder" break, freeing
the new RNA.
5- If the cell has a nucleus, the RNA is further processed and then, moves
through the small nuclear pores to the cytoplasm.
-Transcription is the first step leading to gene expression.
-The stretch of DNA transcribed into RNA molecule is called
a transcription unit and encodes at least one gene.
-If the gene transcribed encodes a protein, the result of transcription is
mRNA, which will then, be used to create that protein by the process of
translation.
-Alternatively, the transcribed gene may encode for either rRNA or
tRNA, other components of the protein-assembly process, or other
ribozymes.
-The transcription unit encoding for a protein contains not only the
sequence that will eventually be directly translated into the protein (the
coding sequence) but also regulatory sequences that direct and regulate
the synthesis of that protein.
-The regulatory sequence before (upstream from) the coding sequence is
called the five prime untranslated region (5'UTR), and the sequence
following (downstream from) the coding sequence is called the three
prime untranslated region (3'UTR).
Transcription unit
Promotor region
It formed of certain sequence that are responsible for initiation of
transcription
Prokaryotic promotor
TATAAT (Pribnow box)
TGTTGACA |(Second nucleotide sequence)

Eukaryotic promotor
TATA (Hogness box)
CAAT box 40 bases in between the two boxes

Transcription region
Segment of DNA to be transcribed into RNA

Termination region
sequence of DNA that helps in termination of transcription
Requirement for RNA synthesis
1- ATP, GTP, UTP, CTP (NTP = nucleotide triphosphate)
2- RNA polymerase
Prokaryotic RNA polymerase
Holoenzyme of RNA polymerase formed of core molecule and specific
protein is sigma factor (δ)
Core molecule formed of 2 α and 2 (β and β/)
Sigma factor (δ)
It helps RNA polymerase to recognize the promotor
Function of RNA polymerase
Searching for the initiation site
Detect termination site
1- Interact with activator and inhibitor
2- Unwinding DNA
-Transcription has no proofreading mechanisms, therefore, transcription
has a lower copying fidelity than DNA replication.
-As in DNA replication, DNA is read from 3' → 5' during transcription.
- Meanwhile, the complementary RNA is created from the 5' → 3'
direction which means that its 5' end is created first in base pairing.
-Although DNA is arranged as two antiparallel strands in a double helix,
only one of the two DNA strands, called the template strand, is used for
transcription, because RNA is only single-stranded molecule.
-The other DNA strand is called the coding strand, because its sequence
is the same as the newly created RNA transcript (except for the
substitution of uracil for thymine).
-The use of only the 3' → 5' strand eliminates the need for the Okazaki
fragments seen in DNA replication.
-Transcription is divided into 5 stages: pre-initiation, initiation,
promoter clearance, elongation and termination.
 1- Pre-initiation:
-In eukaryotes, RNA polymerase, and therefore the initiation of
transcription, requires the presence of a core promoter sequence in the
DNA.
-Promoters are regions of DNA that promote transcription and, in
eukaryotes, are found at -30, -75, and -90 base pairs upstream from the
start site of transcription.
-Core promoters are sequences within the promoter that are essential for
transcription initiation.
-RNA polymerase is able to bind to core promoters in the presence of
various specific transcription factors.
-The most common type of core promoter in eukaryotes is a short DNA
sequence known as a TATA box, found 25-30 base pairs upstream from
the start site of transcription.
-The TATA box, as a core promoter, is the binding site for a transcription
factor known as TATA-binding protein (TBP), which is itself a subunit
of another transcription factor, called Transcription Factor II D (TFIID).
Transcription bubble
It is the region that contains RNA polymerase, opened DNA
and newly formed RNA.
RNA polymerase

Template strand Coding strand

Unwinding
Rewinding

RNA Elongation site

Movement of polymerase
-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 preinitiation complex.
-One transcription factor, DNA helicase, has helicase activity and so is
involved in the separating of opposing strands of double-stranded DNA
to provide access to a single-stranded DNA template.
-Other proteins known as activators and repressors, along with any
associated coactivators or corepressors, are responsible for modulating
transcription rate.
-Thus, preinitiation complex contains:
-1. Core Promoter Sequence
-2. Transcription Factors
-3. DNA Helicase
-4. RNA Polymerase
-5. Activators and Repressors
-Transcription initiation is more complex in eukaryotes.
-Eukaryotic RNA polymerase does not directly recognize the core
promoter sequences.
-Instead, a collection of proteins called transcription factors mediate
the binding of RNA polymerase and the initiation of transcription.
-Only after certain transcription factors are attached to the promoter does
the RNA polymerase bind to it.
-The completed assembly of transcription factors and RNA
polymerase bind to the promoter, forming a transcription initiation
complex.
2- Promoter clearance
-After the first bond is synthesized, the RNA polymerase must clear the
promoter.
-There is a tendency to release the RNA transcript and produce
truncated transcripts (abortive initiation) and is common for both
eukaryotes and prokaryotes.
-Abortive initiation continues to occur until the σ factor rearranges,
resulting in the transcription elongation complex (which gives a 35 bp
moving footprint).
-The σ factor is released before 80 nucleotides of mRNA are synthesized.
- Once the transcript reaches approximately 23 nucleotides, it no longer
slips and elongation can occur which is an energy-dependent process.
- Promoter clearance coincides with phosphorylation of serine 5 on the
carboxy terminal domain of RNA Pol in eukaryotes, which is
phosphorylated by TFIIH.
-One strand of the DNA, the template strand (or noncoding strand), is
used as a template for RNA synthesis.
-As transcription proceeds, RNA polymerase traverses the template
strand and uses base pairing complementarity with the DNA template to
create an RNA copy.
-Although RNA polymerase traverses the template strand from 3' → 5',
the coding (non-template) strand and newly-formed RNA can also be
used as reference points, so transcription can be described as occurring 5'
→ 3'.
-This produces an RNA molecule from 5' → 3', an exact copy of the
coding strand (except that thymines are replaced with uracils, and the
nucleotides are composed of a ribose sin its sugar-phosphate backbone).
- Unlike DNA replication, mRNA transcription can involve multiple
RNA polymerases on a single DNA template and multiple rounds of
transcription (amplification of particular mRNA), so many mRNA
molecules can be rapidly produced from a single copy of a gene.
Elongation

Termination
Termination
-Bacteria use two different strategies for transcription termination.
-In Rho-independent transcription termination, RNA transcription stops
when the newly synthesized RNA molecule forms a G-C-rich hairpin
loop followed by a run of Us.
-When the hairpin forms, the mechanical stress breaks the weak U-A
bonds, now filling the DNA-RNA hybrid.
-This pulls the poly-U transcript out of the active site of the RNA
polymerase, in effect, terminating transcription.
-In Rho-dependent type of termination, a protein factor called "Rho"
destabilizes the interaction between the template and the mRNA, thus
releasing the newly synthesized mRNA from the elongation complex.
- In Rho factor independent termination, formation of palindrome
(means the sequence that can be read the in the opposite directions)
5 – GAATTC – 3
3- CTTAAG – 5
- Formation of hair pin, it is self complementary strand of RNA followed
by several U
Transcription termination in eukaryotes is less understood but involves
cleavage of the new transcript.
Antibiotic inhibitor of transcription
1- Rifamycin
Bind to β subunit of RNA polymerase so prevent transcription
2- Actinomycin D
Bind to DNA template so prevent RNA polymerase on movement on
DNA

Differences between DNA polymerase and RNA polymerase

RNA polymerase DNA polymerase
- Synthesize RNA - Synthesize DNA
- Not need - Needs RNA primer
- No proof reading - Has proof reading activity
- Low fidelity (no nuclease activity) - High fidelity
- Make unwinding - Cannot make unwinding
- Can start transcription denovo alone - Can not start replication alone
- Synthesize RNA only - Synthesize and repair DNA
- One type in bacteria - Three types in bacteria
Reverse transcription

-Some viruses (such as HIV, the cause of AIDS), have the ability to
transcribe RNA into DNA.
-HIV has an RNA genome that is duplicated into DNA.
-The resulting DNA can be merged with the DNA genome of the host
cell.
-The main enzyme responsible for synthesis of DNA from an RNA
template is called reverse transcriptase.
-In the case of HIV, reverse transcriptase is responsible for synthesizing a
complementary DNA strand (cDNA) to the viral RNA genome.
-An associated enzyme, ribonuclease H, digests the RNA strand, and reverse
transcriptase synthesizes a complementary strand of DNA to form a double helix
DNA structure.
-This cDNA is integrated into the host cell's genome via another enzyme
(integrase) causing the host cell to generate viral proteins that reassemble into new
viral particles.
-However, in retroviruses, the host cell remains intact as the virus buds out of the
cell but in the case of HIV, the host cell undergoes apoptosis.
-Some eukaryotic cells contain an enzyme with reverse transcription activity called
telomerase.
-Telomerase is a reverse transcriptase that lengthens the ends of linear
chromosomes.
-Telomerase carries an RNA template from which it synthesizes DNA repeating
sequence, or "junk" DNA.
-This repeated sequence of DNA is important because, every time a linear
chromosome is duplicated, it is shortened in length.
-Telomerase is often activated in cancer cells to enable cancer cells to duplicate
their genomes indefinitely without losing important protein-coding DNA sequence.
-Activation of telomerase could be part of the process that allows cancer cells to
become immortal.
Classes of RNA Polymerases
-In prokaryotic cells, all 3 RNA classes are synthesized by a single
polymerase.
-In eukaryotic cells there are 3 distinct classes of RNA polymerase, RNA
polymerase (pol) I, II and III.
-Each polymerase is responsible for the synthesis of a different class of
RNA.
-The identification of which polymerase synthesizes which class of
RNAs was reported.
-RNA pol I is responsible for rRNA synthesis (excluding the 5S rRNA).
-There are 4 major rRNAs in eukaryotic cells designated by there
sedimentation size.
-The 28S, 5S, 5.8S RNAs are associated with the large ribosomal
subunit and the 18S rRNA is associated with the small ribosomal
subunit.
-RNA pol II synthesizes the mRNAs and some of the small nuclear
RNAs (snRNAs) involved in RNA splicing.
-RNA pol III synthesizes the tRNAs, the 5S rRNA and some snRNAs.
Posttranscriptional Processing of RNAs:

- Bacterial rRNAs and tRNAs undergoes no additional processing, after

being transcribed they are immediately ready for use in translation.
-Translation of bacterial mRNAs can begin even before transcription is
completed due to the lack of the nuclear-cytoplasmic separation that
exists in eukaryotes and to afford a unique opportunity for regulating the
transcription of certain genes.
-An additional feature of bacterial mRNAs is that most are polycistronic
which means that multiple polypeptides can be synthesized from a single
primary transcript.
-Polycistronic mRNAs are very rare in eukaryotic cells but have been
identified.
-In addition, several viruses encode polycistronic RNAs.
-In contrast to bacterial transcripts, eukaryotic RNAs (all 3 classes)
undergo significant post-transcriptional processing.
-All 3 classes of RNA are transcribed from genes that contain introns.
-The sequences encoded by the intronic DNA must be removed from the
primary transcript prior to the RNAs being biologically active.
-The process of intron removal is called RNA splicing, additional
processing occurs to mRNAs, the 5' end of all eukaryotic mRNAs are
capped with a unique 5'—>5' linkage to a 7-methyl guanosine residue.
-The capped end of the mRNA is thus, protected from exonucleases and
more importantly is recognized by specific proteins of the translational
machinery.
-The capping process occurs after the newly synthesizing mRNA is
around 20–30 bases long.
Structure of the 5'-Cap of eukaryotic mRNAs
-Messenger RNAs also are polyadenylated at the 3' end.
-A specific sequence, AAUAAA, is recognized by the endonuclease
activity of by polyadenylate polymerase which cleaves the primary
transcript approximately 11–30 bases 3' of the sequence element.
-A stretch of 20–250 A residues is then added to the 3' end by the
polyadenylate polymerase activity.

Processes of polyadenylation
Splicing of RNAs
-There are several different classes of reactions involved in intron
removal.
-The 2 most common are the group I and group II introns.
- Group I introns are found in nuclear, mitochondrial and chloroplast
rRNA genes, group II in mitochondrial and chloroplast mRNA
genes.
-Many of the group I and group II introns are self-splicing.
-Group I introns require an external guanosine nucleotide as a
cofactor.
-The 3'–OH of the guanosine nucleotide acts as a nucleophile to attack
the 5'–phosphate of the 5' nucleotide of the intron.
-The resultant 3'–OH at the 3' end of the 5' exon then attacks the 5'
nucleotide of the 3' exon releasing the intron and covalently attaching the
two exons together.
-The 3' end of the 5' exon is termed the splice donor site and the 5' end
of the 3' exon is termed the splice acceptor site.
Splicing by Group 1 Introns
-Group II introns are spliced similarly except that instead of an external
nucleophile, the 2'–OH of an adenine residue within the intron is the
nucleophile.
-This residue attacks the 3' nucleotide of the 5' exon forming an internal
loop called a lariat structure.
-The 3' end of the 5' exon then attacks the 5' end of the 3' exon as in
group I splicing releasing the intron and covalently attaching the two
exons together.

Splicing by Group 2 Introns

-The third class of introns is also the largest class found in nuclear
mRNAs.
-This class of introns undergoes a splicing reaction similar to group II
introns in that an internal lariat structure is formed.
-However, the splicing is catalyzed by specialized RNA–protein
complexes called small nuclear ribonucleoprotein particles (snRNPs,
pronounced snurps).
-The RNAs found in snRNPs are identified as U1, U2, U4, U5 and U6.
-Analysis of a large number of mRNA genes has led to the identification
of highly conserved consensus sequences at the 5' and 3' ends of
essentially all mRNA introns.
-The U1 RNA has sequences that are complimentary to sequences near
the 5' end of the intron.
-The binding of U1 RNA distinguishes the GU at the 5' end of the intron
from other randomly placed GU sequences in mRNAs.
-The U2 RNA also recognizes sequences in the intron, in this case near
the 3' end.
-The addition of U4, U5 and U6 RNAs forms a complex identified as
the spliceosome that then removes the intron and joins the two exons
together.
-An additional mechanism of intron removal is the process of tRNA
splicing.
-These introns are spliced by a specific splicing endonuclease that
involves a cut-and-paste mechanism.
- In order for tRNA intron removal to occur the tRNA must first be
properly folded into its characteristic cloverleaf shape.
-Misfolded precursor tRNAs are not processed which allows the splicing
reaction to serve as a control step in the generation of mature tRNAs.
Modifications of tRNA
1-Removal of 5 extrasequence 2- Addition of CCA at 3 end
3-Methyaltion of some bases 4- Addition of anticodon loop
Addition of CCA

Removal of intron
Processing of rRNA
It includes:
5 S rRNA+5.8 S rRNA+28 S rRNA+50 polypeptide chains = 60 S rRNA
18 S rRNA + 30 polypeptide chains = 40 S rRNA
Difference between cytoplasmic mRNA and nuclear analogue
Nuclear mRNA Cytoplasmic mRNA
1- No Post transcription modification 1- Post transcription modification
2- Contain exon and intron 2- Contains exons only
3- No Cap and tail 3- Contains Cap and tail

Chromosomal recombination (translocation)

- It is the movement of genetic material from one arm to the arm of
chromosome which leads to mutation and cancer
Ex. Chronic leukemia there is translocation between chromosome 9 , 22
 Difference between transcription in prokaryote and
eukaryote
Prokaryote Eukaryote

Post transcriptional No Present

modifications
T half Short Long
Transcription and Occur at the Separate
translation same time
RNA Polycistronic Monocistronic
Regulatory region Contains basal Contains basal
region region and
regulatory region
RNA Processing: pre-mRNA → mRNA