1. Berg • Tymoczko • Stryer
Biochemistry
Sixth Edition
Chapter 29
RNA Synthesis and Processing
Part I: RNA synthesis
Copyright © 2007 by W. H. Freeman and Company

2. RNA synthesis is a key
step in the expression of
genetic information
Christmas tree:
Active transcription

3. RNA synthesis, or transcription, is catalyzed
by RNA polymerase
Structural conservation
Mg2+ ion
(more complex regulation)

5. RNA synthesis
(or biological polymerization):
initiation, elongation, and termination
RNA polymerase’s functions:
Search “promoters” (initiation sites;
or cis-acting elements)
Unwind short stretch of dsDNA
Formation of phophodiester bonds
(using rNTP)  processivity
Detect termination signals
Interact with activator and repressor
(transcription factors or trans-acting
factors)  regulation of txn rate

6. RNA synthesis for mRNA, tRNA, and rRNA:
Common in chemistry and steps
Different: RNA polymerase, regulation, and
post-transcriptional processing

8. Transcription in E coli
(holoenzyme)
Core enzyme (contains catalytic site)
s: finds promoter, initiates txn, then dissociates

9. RNA polymerase active sites:
Similar to DNA pol. but
overall structure is dif.

10. Transcription initiation:
Identification of promoter sites
footprinting technique

12. Two common motifs are present on the 5’ (upstream)
side of the transcription start site
-35 sequence and -10 sequence
(consensus seq.)
Core promoter

14. Two common motifs are present on the 5’ (upstream)
side of the transcription start site
-35 sequence and -10 sequence
(consensus seq.)
Core promoter
Start site = +1 (+2…, -1, -2, etc.)
Template strand vs. coding strand
antisense (-) sense (+)

15. Transcription initiation:
promoter activity (or efficiency)
1. strong promoter vs. weak promoter
(in relation to consensus seq.)
2. Distance between two conserved seq.
Other factors:
* Promoter-binding proteins, or
polymerase-binding proteins
* UP element (upstream): -40~-60 of
highly expressed genes (for a subunit)

16. Transcription initiation: recognition of promoter sites  s subunit
Help RNA pol. bind
core promoter
Transcription initiation:
recognition of promoter sites
 s subunit

17. Transcription initiation:
Search for promoter sites
* Holoenzyme slides along dsDNA
* Promoter is encountered by a random
walk in one dimension
* s subunit dissociates when nascent RNA
chain = 9-10 nt
* s subunit next assists another initiation

18. There are multiple types of s subunit
s32: recognize heat-shock genes s54: responds to nitrogen starvation
 s determines where txn starts

19. Transcription initiation: Template unwinding
Closed promoter complex  open promoter complex:
Important step in txn initiation

20. Negative supercoiling facilitates unwinding
Exception: promoter of Topoisomerase II gene
 negative supercoils decreases txn
(negative feedback)
Supercoils: change structual relation of
the –10 and –35 regions

21. RNA synthesis can start de novo
First base at 5’end: pppG or pppA

22. RNA synthesis grow in the 5’-3’ direction

23. Transcription elongation: Txn bubble on DNA template
Elongation: 1. formation of first bond
2. dissociation of s  strong binding of core
3. 50 nt/sec
txn bubble
8bp

24. Surface model of a bubble
“melted” DNA
Surface model of a bubble

25. RNA-DNA hybrid
separation by RNA pol.
Fixed structure of
the txn bubble

26. Transcription elongation:
Fidelity of the RNA pol.
Contains proofreading nuclease activity
104 – 105
(DNA polymerase: 107)
Higher error rate can be tolerated:
*Mistakes are not transmitted
*Many RNA transcripts for most genes

27. Transcription termination:
Formation of phosphodiester bond stops
RNA-DNA hybrid dissociates
Melted DNA rewinds
RNA pol. releases DNA
Transcribed region of DNA
contains top signal

28. Palindromic GC-rich region followed by AT-rich
In DNA:
Palindromic GC-rich region
followed by AT-rich
Stable hairpin because
of GC-rich
Txn stop

29. How does this structure terminate transcription?
RNA pol. pauses after such structure
rU-dA: highly unstable
Weakly bound nascent RNA dissociates

30. Transcription termination:
Additional factor, rho (r)
How does r termination of RNA synthesis?

31. 72 nt Hexameric rho is ATPase in the presence of ssRNA
Activated by C-rich &
G-poor RNA region
Hydrolysis
RNA-DNA helicase:
breaking hybrid helix
72 nt

32. Transcription termination:
Additional factor, nusA protein
binds specific termination signals in E. coli:
“Attenuators”
Important feature of the termination
mechanism: signals lie in RNA (not DNA)

33. Antibiotics: inhibitors of transcription
Example no. 1

34. Rifampicin blocks txn initiation
Binds a pocket occupied by newly
formed DNA-RNA hybrid
 Competitor
Conserved in prok. not in euk.  antibiotic

35. Example no. 2: actinomycin D
* Binds specifically and tightly to dsDNA
* Intercalation: between neighboring base-pair
* at low concentrations, inhibit txn but not
DNA replication (both prok. and euk.)

36. Eukaryotic transcription: more complex regulation
Differential txn regulation  cell types

38. Three keys in euk.:
Nuclear membrane
Txn regulation:
more promoters,
enhancer
RNA processing:
ex. splicing

39. Three different types of euk. RNA polymerases
* Large proteins
* 8-14 subunits
Similarity:
RNA pol. II: 220-kd (largest) subunit has a
carboxyl-terminal domain (CTD)
 YSPTSPS repeats
 serine phosphorylation

40. Binds to pol. II tightly and inhibits elongation
(action is conserved in euk.)

41. Euk. transcriptional initiation: promoters
Conserved sequences for pol. binding
Different in sequence and position
RNA polymerase-dependent

42. Promoter binding proteins
RNA polymerase I: rDNA gene
X hundreds
TATA-like seq.:
Ribosomal initiator element
Promoter binding proteins
RNA pol. I

43. RNA polymerase II a set of conserved elements: combination
Enhancer: unique to euk., more than 1 kb from promoter

44. RNA polymerase III: intergenic
(within transcribed region)

45. RNA polymerase II promoter: 3 common elements
Mutagenesis exp’ts, footprinting, and sequence comparisons 1 2
Initiator element (-3 ~ +5) 3
Downstream core
promoter element
(+28 ~ +32)

46. -30 ~ -100
(similar to prok. –10)

47. On template (antisense) strand
-40 ~ -150 (similar to prok. –35)
On template (antisense) strand
 Constitutively expressed genes
Euk. promoter elements: recognized by txn factors

49. Txn initiation: TFII’s TFIID to TATA box (seq. specific)
Basal txn apparatus
Binds “mediator”

50. TBP and TATA-box DNA * Unwinding (minor groove)
* Hydrophobic interaction
* Phenylalanine intercalation
* Asymmetry

52. initation  elongation
(by TFIIH)
initation  elongation
* Stabilize txn elongation
* Recruit RNA-processing enzymes

53. Multiple transcription factors bind euk. promoters
* Basal txn apparatus: minimal transcription (low)
* Additional txn factors: bind other sites
for high txn rate
* Upstream stimulatory sites: variable
(sequence & location)

54. There are many transcription factors
Ex. heat-shock transcription factor (HSTF)
bind directly to consensus seq.
in response to high temperature

55. Another level of promoter activity: enhancers
* Have no promoter activity
* Act from several thousand bp away
* Can be downstream, upstream, or intragenic
* Cell type-specific
In yeast: upstream activator sequences (UASs)
txn factors and cis-acting elements:
key to txn regulation
transcription: key to gene expression