This slide is primarily for undergraduate students in medicine and basic sciences.

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

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3. Friday, December 19, 20143 Rajesh Chaudhary

4. DNA Vs RNA
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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

5. Friday, December 19, 20145 Rajesh Chaudhary
Three different RNA differ from each other in terms of size,
function and concentration.

6. 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)
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Rajesh Chaudhary

7. 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

8. 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

9. 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

10. 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.

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

12. 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

13. 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 !

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

15. Elongation in transcription
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16. Elongation in transcription
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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

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1. Transcription is regulated at
several steps
Rajesh Chaudhary

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

19. 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?

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s cycle

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

22. Transcription in Eukaryotes
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Rajesh Chaudhary

23. 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

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

25. 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)

26. 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

27. 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

28. 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”.

29. 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

30. Possible locations of enhancer
sequence
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Inhibitors of RNA polymerase II
 a-amanitin of Amanita phalloides
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31. 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

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

33. Post transcriptional modification of
tRNA
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nucleotidyltransferase
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34. 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

35. References
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Rajesh Chaudhary