Transcript Lecture 6 -Transcription 2

Biochemistry 201
Biological Regulatory Mechanisms
January 24, 2013
Mechanism of transcription elongation
References
I.General
Chapter 12 of Molecular Biology of the Gene 6th Edition (2008) by Watson, JD, Baker, TA, Bell, SP, Gann, A, Levine, M,
Losick, R. 377-414.
II.Evolution
Werner, F. and Grohmann, D. Evolution of multisubunit RNA polymerases in the three domains of life. (2011) Nature
Rev. Microbiol 9: 85-98
Lane WJ, Darst SA.(2009) Molecular Evolution of Multisubunit RNA Polymerases: Sequence Analysis.J Mol Biol. 2009
Nov 3. [Epub ahead of print]PMID: 19895820 [PubMed - as supplied by publisher]
II. A few of the many insights from RNA polymerase structures
Cramer, P. (2002) Multisubunit RNA polymerases. Curr Opin Struct Biol 12:89-97.
Murakami KS, Darst SA. (2003) Bacterial RNA polymerases: the holo story. Curr Opin Struct Biol 13:31-9.
*Cramer, P. (2004) RNA polymerase II structure: from core to functional complexes. Curr Opin Genet Dev 14:218-26.
Review.
Wang, D. Bushnell DA, Westover KD, Kaplan, CD, Kornberg RD. Structural basis of transcription: role of the trigger loop
in substrate specificity and catalysis. Cell. 2006 Dec 1;127(5):941-54.
Cramer, P. (2007). Gene transcription: extending the message. Nature, 448(7150), 142-3.
*
*Vassylyev, DG, Vassylyeva, MN, Zhang, J, Landick, R (2007). Structural basis for substrate loading in bacterial RNA polymerase.
Nature, 448(7150), 163-8.
IV. Proofreading
*Zenkin, N, Yuzenkova, y Severinov K Transcript-assisted transcriptional proofreading.
Science. 2006 Jul 28;313(5786):518-20
Sydow JF, Cramer P. (2009) RNA polymerase fidelity and transcriptional proofreading.Curr Opin Struct Biol. 2009 Dec;19(6):732-9.
Epub 2009 Nov 13.
Sydow JF, Brueckner F, Cheung AC, Damsma GE, Dengl S, Lehmann E, Vassylyev D, Cramer P.(2009) Structural basis of
transcription: mismatch-specific fidelity mechanisms and paused RNA polymerase II with frayed RNA. Mol Cell. Jun 26;34(6):71021.
V. Pausing
Artsimovitch, I. and Landick, R (2000). Pausing by bacterial RNA polymerase is mediated by mechanistically distinct classes of
signals. PNAS 97: 7090-7095
Zhang J, Palangat M, Landick R. Role of the RNA polymerase trigger loop in catalysis and pausing. Nat Struct Mol Biol. 2010
Jan;17(1):99-104. Epub 2009 Dec 6.
*Shaevitz, j. Abbondanzieri E, Landick R. and Block S (2003) Backtracking by single RNA polymerase molecules observed at near
base pair resolution. Nature 426: 684-687
Herbert, K., La Porta, A, Wong B, Mooney, R. Neuman, K. Landick, R. and Block, S.(2006). Sequence-Resolved Detection of
Pausing by Single RNA Polymerase Molecules. Cell 125:1083-1094
*Weixlbaumer, A, Leon, K, Landick, R and Darst SA (2013) Structural basis of transcriptional pausing in bacteria. Cell, in press
VI. Regulation through the 2˚ channel
Paul BJ, Barker MM, Ross W, Schneider DA, Webb C, Foster JW, Gourse RL. (2004) DksA: a critical component of the
transcription initiation machinery that potentiates the regulation of rRNA promoters by ppGpp and the initiating NTP.
Cell. 6:311-22.
Important Points
1. Cellular RNA polymerases have no structural similarities to DNA polymerases; even though they carry
out similar reactions, they are a separate evolutionary invention.
2. Cellular RNA polymerases have many moving parts. For example, incoming NTPs first base pair with
the template in a catalytically inactive form and are subsequently pushed into the active site by folding of
the “trigger loop”. This movement links correct nucleotide recognition to catalysis and thereby increases
fidelity. In other words, the polymerase takes two looks at the incoming NTP.
3. The active site of cellular RNA polymerases can be regulated by accessory proteins that penetrate the
secondary channel (also called the pore), position a Mg ion, and thereby cause the active site to cleave
RNA rather than polymerize it. This reaction is not simply the reverse of the polymerization reaction.
4. RNA proofreading occurs when a mispaired nucleotide positions a Mg at the active site, stimulating
cleavage reaction.
5. Transcriptional pauses are integral to the transcription process and are integral to transcriptional
regulation.
Comparison of transcription and replication
Replication
Speed
500 nucs/sec: bacteria
50 nucs/sec: euks
Error rate 1/109(including
mismatch repair)
Job
Copy every sequence in
the genome once
Transcription
10-30 nucs/sec
1/104- 1/105
Transcribe segments
of the genome at
highly variable rates
RNA polymerases vs. DNA polymerases
Similarities
1. Polymerize NTPs using DNA as template
2. Similar reaction mechanism
3. Both remove errors
Differences
1. Ribonucleoside vs deoxyribonucleside triphosphates
2. No structural similarity
3. RNAP initiates de novo; DNAP elongates prexisting chains
4. RNAP active site does both NTP addition and proofreading
5. Active site of RNAP is highly regulated, enabling a
dynamic response to signals during elongation
Nucleotide Addition Cycle (NAC) and Definition of states
Steps in the Nucleotide Addition Cycle ( NAC)
NTP addition
rate limiting step
pretranslocated
Post-translocated
NTP bound
Steps in the Nucleotide Addition Cycle ( NAC)
pretranslocated
Post-translocated
NTP addition
Backtracked
RNA polymerase structure revisited: An Elongation Perspective
Structure of RNAP
Cutaway view of
elongating complex
Clamp and nucleic acids: the
switch region controls clamp
opening and is a target of the
antibiotic myxopyronin
Concept of a tuneable active site:
Optimized for diversity of response not speed
1. Reactions at active site
2. Mechanism of Nucleotide addition
3. RNA cleavage; extrinsic proofreading
4. RNA cleavage-Intrinsic proofreading
5. Pausing
(1) The Active Site catalyzes two distinct reactions
Nucleotidyl addition (RNA polymerization):
A nucleoside monophosphate from a templated NTP
substrate) is attached to 3’OH of growing RNA
chain; PPi released.
Mg++ (A) binds RNAP; Mg++ (B) binds incoming NTP
(1) The Active Site catalyzes two distinct reactions
Nucleotidyl addition (RNA polymerization):
A nucleoside monophosphate from a templated NTP
substrate) is attached to 3’OH of growing RNA
chain; PPi released.
Mg++ (A) binds RNAP; Mg++ (B) binds incoming NTP
Hydrolysis (RNA cleavage, proofreading)
Uses OH- as nucleophile to cleave transcript when an
internal phosphodiester bond occupies the active
site; mediated by RNAP itself; accelerated by
“cleavage factors” that bind in the 2˚ channel
Mg++ (A) binds RNAP; Mg++ (B) binds RNA chain or
cleavage factor(GreA/B; TFIIS)
Both reactions use a two-Mg2+-mediated bimolecular
nucleophilic substitution (SN2) reaction mechanism
(2) Structure of the elongation complex
“Frozen” elongating complexes can be assembled on a nucleic acid scaffold
Complexes were used to determine RNAP structure during nucleotide addition
Determined two structures of elongating RNA polymerase
a) Elongation complex with non-hydrolyzable NTP
b) Elongation complex with non-hydrolyzable NTP
and streptolydigin ( elongation inhibitor)
RNA-P looks at each incoming NTP twice before addition
Substrate enters through 2˚ channel
NTP binds at “preinsertion site”
usingW-C base pairing; RNAP
contacts discriminate NTP /dNTP;
2nd Mg++ too far for catalysis
(structure in the presence of NTP and
streptolydigin or -amanitin)
Trigger-loop folds and forms 3-helix
bundle with bridge helix; active center
closes allowing additional check for
complementarity; 2˚ channel constricts
(structure in the presence of NTP)
Incorporation of mononucleotide and
release of pyrophosphate
The trigger loop is a key moving part of RNA
polymerase; its folding is required for nucleotide
addition
The Cleavage Reaction
(3) The Transcript Cleavage Reaction
Transcript cleavage factors bind in the 2˚ channel; a Mg++
bound to the tip mediates cleavage of a “backtracked” RNA
Misincorporated NTPs promote backtracking; transcript cleavage factors
promote error correction (cleavage factors also promote elongation)
However, RNAP alone can also correct
errors. What is the mechanism?
(4) Demonstration of intrinsic proofreading by RNAP in vitro
A. Assemble properly paired or mismatched 5’ labeled transcript on a scaffold
B. Add Mg++ , denature, run on denaturing gel, autoradiograph
Predictions
Results
(5) Transcriptional pauses
Transcriptional pauses
are really important
Coordinate transcription (RNAP movement) with:
1) Folding nascent RNA
2) Other RNA processes
translation, degradation, export, splicing
3) Regulator binding (TAR—HIV; RfaH prokaryotes)
Promoter proximal pauses poise RNAPII for gene expression
in metazoans
Current view of Pausing
X
X
(?)
Elementary Pause Complex
Current view of Pausing
(?)
Elemental Pause Elongation Complex
X
X
Current view of Pausing
(?)
Elemental Pause Elongation Complex
How to measure pauses
Time (Min)
Run-off transcript--
Pause transcript--
Pauses are characterized by duration and
“efficiency” (probability of entering the
pause state at kinetic branch between
pausing and active elongation)
Aliquots of a synchronized, radiolabeled, single-round transcription assay were removed at various times and
electrophoresed on a polyacrylamide gel; separation by size
Elements of a hairpin stabilized pause.
Elements of a back-tracked pause
1.
Enabled by ability of RNA to translocate relative to the DNA
template; when there is a less stable DNA/RNA hybrid, tendency of
RNA is to backtrack until a more stable RNA/DNA hybrid is achieved
2. Backtrack pauses are reduced by creating a more stable RNA/DNA
hybrid, or by addition of GreA (promotes transcript cleavage and
realignment of active center
3. Position of RNA polymerase on DNA can be determined by
footprinting using exonuclease III (degrades DNA from 3’end)
Pausing can also be measured using single molecule
techniques
Can follow single molecules over long times and detect very short pauses
Identification of Elemental pauses
Trace of two RNA polymerase
molecules
Backtracking by eye:
phase 1 (backtracking, solid line)
phase 2 (pause, dotted line)
phase 3 (recovery, solid line).
Representative short pause (3 s);
No backtracking
*Short pauses account for 95% of all pausing events; subsequent studies
confirmed that they are not backtracked and occur at specific sequences
(ubiquitous/elemental pauses)
Determining the structure of paused RNAP
1. Create an nucleic acid scaffold ending 2 nt prior to an expected elementary pause
2. Monitor addition of the 2 NTPs to assure expected pause is observed
3. Determine structure of paused RNAP, as well as one from a
comparable scaffold with no pausing
Clamp opening in paused complex disrupts BH/TL
contacts to clamp anchor (switches 1&2) and inhibits
TL folding
Darst, in press Cell
Current view of Pausing
(?)
Elemental Pause Elongation Complex
NusG, the only universal elongation factor,
exhibits divergent interactions with other regulators
NusG-like NTD binds across the cleft in all three
kingdoms of life, apparently locking the clamp
against movements (& encircling DNA)
adapted from Martinez-Rucobo et al. 2011 EMBO J. 30:1302