Transcription

TRANSCRIPTION
Department of Biochemistry, Kathmandu Medical CollegeFriday, December
19, 2014
1Rajesh Chaudhary

Friday, December 19, 20142 Rajesh Chaudhary

DNA Vs RNA
Friday, December 19, 2014Rajesh Chaudhary
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Like DNA, RNA are unbranched polymeric molecules composed of
nucleoside monophosphate joined together by phosphodiester bonds.
DNA RNA
Usually double stranded. Usually single stranded.
Size: Bigger than RNA Smaller than DNA
Bases: A, G, C, T A, G, C, U

Three different RNA differ from each other in terms of size,
function and concentration.

Introduction of RNA
 How is genetic information is executed?
 Types of RNA
 mRNA
 tRNA
 rRNA
 Does our body have just these RNA or some more?
 What is the role of RNA? (Structural, Catalytic)
Friday, December 19, 2014
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Rajesh Chaudhary

Transcription
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 Catalytic RNAs are also known as RIBOZYMES.
 What is the functional difference between DNA and
RNA?
 What are the differences and similarities between
replication and transcription?
 Entire structure or part of structure?
Rajesh Chaudhary

Transcription / RNA synthesis
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 Similarities
 Fundamental chemical mechanism
 Its polarity (direction of synthesis)
 Use of template
 Similarities in phages (initiation, elongation and termination)
 Differences
 Use of primer
 Segment
 What is cellular transcriptome?
Rajesh Chaudhary

Transcription
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 DNA template is copied in the 3’  5’ direction
 Synthesis of new RNA molecule = 5’  3’
Process of transcription
Initiation Elongation Termination
Rajesh Chaudhary

Properties of prokaryotic RNA
polymerase
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 One species of RNA synthesize all of the RNA except small RNA that
is needed for DNA replication.
 … is a multisubunit enzyme that recognizes the sequence of DNA.
 Makes RNA copy of DNA.
 New RNA is synthesized in 5’3’ direction – antiparallel to its DNA
template.
 For one gene, only one template of dsDNA functions as template.
 RNA polymerase involves cores enzyme and auxillary enzymes.

RNA polymerase
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 Core enzyme Vs
Holoenzyme
 Termination factor r
Rajesh Chaudhary
Different sigma factor recognizes different genes.

Steps in RNA synthesis
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 How long is a transcription unit? (I.e. Its extension)
 What is the name of the product formed after
transcription?
 Initiation
 RNA binding at promoter
Rajesh Chaudhary

Initiation of transcription
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 Pribnow box
 -35 sequence
Rajesh Chaudhary
Mutation in either the Pribnow box or the -35 sequence can affect the
transcription of the gene controlled by mutant promoter !

Elongation in transcription
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The functional differences: “Topoisomerase I” Vs “Topoisomerase II”

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 Supercoils are disentangled by topoisomerase I and II.
 RNA polymerase synthesizes short transcript (<11 nucleotides)
of DNA sequence.
 Elongation begins when transcript is over 10 nucleotide in
length.
 RNA polymerase has no known proofreading function.
1. How fast nucleotides are added during the elongation step?
2. What is the error rate in transcription process?
Rajesh Chaudhary

1. Transcription is regulated at
several steps
Rajesh Chaudhary

Termination of transcription
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Termination
r-independent r-dependent
Rajesh Chaudhary

r-dependent termination
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 r-factor: hexameric ATPase with helicase activity
 Binds C-rich “rho” recognition site near 3’-end of the nascent
RNA using ATPase activity.
 ATP-dependent RNA-DNA helicase activity of r separates the
hybrid  new RNA
What does ATPase do?

s cycle

Action of antibiotics
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 Antibiotics prevent bacterial growth by inhibiting RNA
synthesis.
 E.g.: rifampin, Actinomycin D
Rajesh Chaudhary

Transcription in Eukaryotes
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Transcription in Prokaryotes Vs
Eukaryotes
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 Differences
 In terms of RNA polymerase
 Eukaryotes: Pol I, Pol II and Pol III
a) Pol I: synthesize precursors of rRNA (28S, 18S, 5.8S)
b) Pol II: synthesizes precursors of mRNA
c) Pol III: produces sRNA including tRNA, 5S rRNA and some
Srna.
 Binding of polymerase is affected by enhancer sequence.
 Similarities
 Polymerase, dNTP, 3 steps of transcription
Rajesh Chaudhary

Chromatin structure and gene
expression
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 DNA, histone, nucleosome
and chromatin – how are
they arranged?
Nucleosome
Chromatin
Euchromatin Heterochromatin
Rajesh Chaudhary

Mechanism of chromatin remodeling
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Rajesh Chaudhary
Chromatin
Inactive chromatin Active chromatin
(Acetylation)
Enzyme: histone acetyl transferase
(Deacetylation)
Enzyme: histone deacetylase (restores +ve charge)

Eukaryotic gene promoter consensus
sequences
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Transcription factors recognize DNA through a variety of
DNA-binding motifs, such as zinc fingers and leucin zippers.
Rajesh Chaudhary

Transcription factors
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 What is transcription factors? Why is it necessary?
Different transcription factors for different genes.
 Coactivator: include the histone acetyltransferase
proteins involved in chromatin remodeling.
Rajesh Chaudhary

Transcription factors
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Cis-acting Vs Trans-acting elements ?
Enhancer Vs Silencer
Enhancers contain DNA sequence called “response elements” that bind specific transcription factors called “activators”.

Role of enhancers in eukaryotic gene
regulation
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 Special cis-acting element
 Increases process of transcription by RNA polymerase II
 Located on the same chromosome where gene is
located
 Presence can be upstream (towards 5’-end) or
downstream (towards 3’-end) to the gene
Rajesh Chaudhary

Possible locations of enhancer
sequence
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Inhibitors of RNA polymerase II
 a-amanitin of Amanita phalloides
Rajesh Chaudhary

Post transcriptional modification
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 Both prokaryotic and Eukaryotic tRNA and rRNA is
posttranscriptionally modified.
 Prokaryotic mRNA is generally identical to its primary
transcript while eukaryotic mRNA is extensively modified.
 tRNA are further modified to give a unique identity to each
species.
Rajesh Chaudhary

Post transcriptional modification of
rRNA
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ribonuclease
Rajesh Chaudhary
Eukaryotic 5S RNA is synthesized by Pol III
and modified separately.

Post transcriptional modification of
tRNA
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nucleotidyltransferase
Rajesh Chaudhary

Post-transcriptional modification of
eukaryotic mRNA
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 The collection of all precursor molecules for mRNA are called
“heterogeneous nuclear RNA (hnRNA)”.
 Modification occurs in nucleus.
Modifications
1. 5’-capping
2. Addition of poly-A tail
3. Removal of introns

References
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Rajesh Chaudhary

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