Biochemistry 201Biological Regulatory MechanismsJanuary 24, 2013

Mechanism of transcription elongation

ReferencesI.General

Chapter 12 of Molecular Biology of the Gene 6 th 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):710-21.

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˚ channelPaul 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

Transcription

Speed 500 nucs/sec: bacteria 10-30 nucs/sec 50 nucs/sec: euks

Error rate 1/109(including 1/104- 1/105

mismatch repair)

Job Transcribe segments of the genome at highly variable rates

Copy every sequence inthe genome once

Replication

RNA polymerases vs. DNA polymerases

Similarities1. Polymerize NTPs using DNA as template

2. Similar reaction mechanism

3. Both remove errors

Differences1. Ribonucleoside vs deoxyribonucleside triphosphates 2. No structural similarity

3. RNAP initiates de novo; DNAP elongates prexisting chains

5. Active site of RNAP is highly regulated, enabling a dynamic response to signals during elongation

4. RNAP active site does both NTP addition and proofreading

Nucleotide Addition Cycle (NAC) and Definition of states

Steps in the Nucleotide Addition Cycle ( NAC)

pretranslocated

Post-translocated

NTP bound

NTP a

dd

itio

n

rate

lim

itin

g s

tep

Steps in the Nucleotide Addition Cycle ( NAC)

pretranslocated

Post-translocated

NTP addition

Backtracked

RNA polymerase structure revisited: An Elongation Perspective

Cutaway view of elongating complex

Clamp and nucleic acids: the switch region controls clamp opening and is a target of the

antibiotic myxopyronin

Structure of RNAP

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

A nucleoside monophosphate from a templated NTP substrate) is attached to 3’OH of growing RNA chain; PPi released.

(1) The Active Site catalyzes two distinct reactions

Nucleotidyl addition (RNA polymerization):

Mg++ (A) binds RNAP; Mg++ (B) binds incoming NTP

A nucleoside monophosphate from a templated NTP substrate) is attached to 3’OH of growing RNA chain; PPi released.

Hydrolysis (RNA cleavage, proofreading)

(1) The Active Site catalyzes two distinct reactions

Both reactions use a two-Mg2+-mediated bimolecular nucleophilic substitution (SN2) reaction mechanism

Nucleotidyl addition (RNA polymerization):

Mg++ (A) binds RNAP; Mg++ (B) binds incoming NTP

Mg++ (A) binds RNAP; Mg++ (B) binds RNA chain or cleavage factor(GreA/B; TFIIS)

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

“Frozen” elongating complexes can be assembled on a nucleic acid scaffold

(2) Structure of the elongation complex

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

Trigger-loop folds and forms 3-helix bundle with bridge helix; active center closes allowing additional check for complementarity; 2˚ channel constricts

Incorporation of mononucleotide and release of pyrophosphate

(structure in the presence of NTP and streptolydigin or -amanitin)

(structure in the presence of NTP)

The trigger loop is a key moving part of RNA polymerase; its folding is required for nucleotide

addition

The Cleavage Reaction

Transcript cleavage factors bind in the 2˚ channel; a Mg++ bound to the tip mediates cleavage of a “backtracked” RNA

However, RNAP alone can also correct errors. What is the mechanism?

(3) The Transcript Cleavage Reaction

Misincorporated NTPs promote backtracking; transcript cleavage factors promote error correction (cleavage factors also promote elongation)

(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:

2) Other RNA processes translation, degradation, export, splicing

1) Folding nascent RNA

3) Regulator binding (TAR—HIV; RfaH prokaryotes)

Promoter proximal pauses poise RNAPII for gene expression in metazoans

Current view of Pausing

(?)

Elementary Pause Complex

XX

Current view of Pausing

(?)

Elemental Pause Elongation Complex

XX

Current view of Pausing

(?)

Elemental Pause Elongation Complex

Aliquots of a synchronized, radiolabeled, single-round transcription assay were removed at various times and electrophoresed on a polyacrylamide gel; separation by size

Time (Min)

Pause transcript--

Run-off transcript--

How to measure pauses

Pauses are characterized by duration and “efficiency” (probability of entering the pause state at kinetic branch between pausing and active elongation)

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 polymerasemolecules

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