Biochemistry 201Biological Regulatory MechanismsJanuary 24, 2013

Transcription elongation and its regulation

References

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.

Role of the RNA Pol II CTD

*McCracken, S, Fong, N, Yankulov, K, et al. (1997). The C-terminal domain of RNA polymerase II couples mRNA processing to transcription. Nature, 385(6614), 357-61.

Tietjen,J. ……Ansari, A. Chemical-genomic dissection of the CTD code (2010) NMSB: 17: 1154-1162

Mayer, A. ….Cramer, P. Uniform transitions of the general Pol II transcription apparatus (2010) NMSB 17:1272-79

Buratowski, S (2009) progression through the RNA polymerase II CTD cycle ( Review). Mol Cell 36: 541-546

Chapman, R… Eick, D. Molecular evolution of the RNA polymerase CTD. Trends in Genetics (2008): Jun;24(6):289-96. Epub 2008 May 9. Review.PMID: 18472177

Elongation Control

BBA2013-- Issue 1874 devoted to reviews of transcription elongation

Zhou Q, Li T, Price DH (2012) RNA polymerase II elongation control .Annu Rev Biochem. 2012;81:119-43.

Rougvie A and Lis JT (1988) The RNA Polymerase II Molecule at the 5’ end of the uninduced hsp70 gene of D. melangaster is transcriptionally engaged. Cell 54: 795-804

Zobeck, KL….Lis Jt (2010) Recruitment timing and dynamics of transcription factors at the Hsp70 Loci in Living Cells Mol Cell 40 965-75

Peterlin, BM and Price DH (2006) Controlling the Elongation Phase of transcription with P-TEFb Mol Cell 23: 297 – 305

Nechaev S…..Adelman K. (2010) Global Analysis of short RNAs reveals widespread Promoter Proximal Stalling and Arrest of Pol II in Drosophila Science 327: 335-38

Gilchrist, DA,……Adelman, K. (2010) Pausing of RNA Polymerase II Disrupts DNA specified Nucleosome Organization to enable precise gene regulation. Cell 143: 540-51Chen, Y.,….Handa, H.(2009) DSIF, the Paf1 complex and Tat-SF1 have nonredundant, cooperative roles in RNA polymerase II elongation. Genees Dev 23: 2765 -77

Liu, Y……Hahn, S. (2009) Phosphorylation of the transcription elongation factor Spt5 by yeast Bur1 kinase stimulates recruitment of the PAF complex. MCB 29: 4852-63

Wu, C-H,….Gilmour, D. ( 2003)NELF and DSIF cause promoter proximal pausing on the Hsp70 promoter in drosophila. Genes Dev 17: 1402-14

Kim, J Guemah M and Roeder, RG.(2010) The human PAD1 Complex Acts in Chromatin Transcription elongation both independently and cooperatively with SII (TFIIS) Cell 140: 491 -503

Genome wide elongation technologiesChurchman LS, Weissman JS. (2011) Nascent transcript sequencing visualizes transcription at nucleotide resolution. Nature 469: 368-73. doi: 10.1038/nature09652. (Net-seq)

Kwak, H… and Lis, JT (2013) Precise maps of RNAP reveal how promoters direct initiation and pausing. Science 339: 950 (PRO-seq)

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 and are extensively used in both prokaryotes and eukaryotes

6. The RNA polymerase CTD is a long series of 7-amino acid repeats. When transcription is initiated, serine 5 of the repeat is phosphorylated by TFIIH. As elongation proceeds, serine 5 is gradually dephosphorylated and serine 2 is gradually phosphorylated by enzymes carried along with the RNA polymerase. This dynamic pattern of modification couples transcription to processing of the newly-synthesized RNA.

7. Promoter proximal pauses ( 20-50 nucleotides) are extensively used for regulation in eukaryotes.

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

Replication vs transcription

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

Cutaway view of elongating complex

Structure of RNAP

Current view of Role of reactions at active site

(?)

Elementary Pause Complex

XX

Current view of Role of reactions at active site

(?)

Elemental Pause Elongation Complex

XX

Current view of Role of reactions at active site

(?)

Elemental Pause Elongation Complex

Transcript cleavage

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

(1) 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)

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)

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

RNAP alone can also correct errors. Here a backtracked RNA chain binds 2nd Mg++ to promote cleavage by the active site

(2) The Transcript Cleavage Reaction

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

(3) 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

The central role of pausing in control

(?)

Elemental Pause Elongation Complex

Transcript cleavage

11 nt from 3’ endof mRNA

~ 8 nt from 3’ end; terminal nts highly enriched in U’s; A-U bp are very weak

AT rich;misincorporation

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)

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)

Pauses can also be measured using single molecule technology

Pauses can also be measured genome wide using NET-seq

Matt Larson ( Weissman lab)

Regulating Termination: Attenuation control

3. RNA polymerase pausing is critical for this regulatory mechanism

2. External inputs can alter the equilibrium between mRNA states

1. Stabilizing alternative 2˚structures of mRNA can lead to either elongation or termination

Case study: Use of a Pause hairpin in “Attenuation” at the trp operon

Low Trp

High Trp

2:3 is an “antiterminator”hairpin

1:2 is a pause hairpin3:4 is an intrinsic terminator

Leader peptide has tryptophan residues

Regulated “attenuation” (termination) is widespread

Switch between the “antitermination” and “termination”Stem-loop structures can be mediated by:

1. Ribosome pausing ( reflects level of a particular charged tRNA): regulates expression of amino acid biosynthetic operons in gram - bacteria

2. Uncharged tRNA: promotes anti-termination stem-loop in amino acyl tRNA synthetase genes in gm + bacteria

3. Proteins: stabilize either antitermination or termination stem-loop structures

4. Small molecules: aka riboswitches

5. Alternative 2˚ structures can also alter translation, self splicing, degradation

NusG, the only universal elongation factor, exhibits divergent interactions with other

regulators

E. coli NusG: A 21kD essential elongation factor

NTD CTD

Activities: 1. Increases elongation rate 2. suppresses backtracking 3. Required for anti-termination mechanisms 4. Enhances termination mediated by the rho-factor

How does one 21Kd protein mediate all of these activities?

NGN domain KOW domain

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

The N-terminal NGN domain increases elongation and decreases pausing

The CTD of NusG interacts with other protein partners

CTDNusE is part of a complex of proteins mediating antitermination/termination depending on its protein partnersNusE

50 µM

10 nM

RhoRho is an RNA binding hexamer that mediates termination by dissociating RNA from its complex with RNA polymerase and DNA using stepwise physical forces on the RNA derived from alternating protein conformations coupled to ATP hydrolysis

Although the CTD mediates the protein interactions involved in termination and antitermination, full length NusG is required for both processes, presumably because NusG must be tethered to RNA polymerase for these functions

Coupled syntheses.

J W Roberts Science 2010;328:436-437

Published by AAAS

Elongation control in eukaryotes

The RNA Polymerase II CTD (or tail)

Regions upstream (R1) and downstream (R3) of the heptad repeat region are enriched in the submotifsProline can be cis or -trans

YSPTSPSP P P

5 repeats in plasmodium 26 repeats in yeast52 repeats in mammals

Heptad repeat unit

2 5 7

5 - amaR CTD

Mouse RNA Pol II

wt

52

What is the role of the Pol II CTD?

examine RNAs

50 hrs.

HeLacells Introduce

CTD construct - amanitin

Splicing, processing of 3’ end, termination were all affected

Nature 385: 357 (1997)

How the Polymerase CTD Couples Transcription to other processes

YSPTSPS

Pcapping factorsTF II H,

Mediator

elongation

YSPTSPS

P P

YSPTSPS

P

3’ end processing factors

splicing components histone methylase DNA repair enzymes

Further elongation

phosphatases (Rtr1(2?)

pTEFb

(Cdk9)

Kinase/ phosphataseFactors recruited

In S. cerevisiae, shared by Cdk1 and Bur 1

YSPTSPS Mediator, activators, GTFs

Phosphatases (Fcp1, ssu72)Termination

2 5 7

TECs are community organizers

The major steps in mRNA processing (trx, 5’ capping, polyA addition, splicing) all occur together on a transcript extruded from the exit channel of RNAP although they can be reconstituted independently in vivo Principles of “cotranscriptionality” to integrate nuclear metabolism1.Permits coupling between different biogenesis steps; eg crosstalk; suspected when decreasing one step has effects on 2 nd; could always be indirect

a. Landing pad—increase concentration of reactants—proteins involved in capping etcb. Allosteary: guanosyl transferase of capping enzyme activated by interaction with phosphorylated CTDc. Kinetic coupling—optimize timing

 2. Impose order or control

a. Juxtaposition of proteins permits assembly, competitive interactions handoffs; often mutually exclusive PPisb. directions emanating from phopshorylation state of CTD

 3. A locator for nuclear machines –DNA repair, modification etc

Bentley: Cotranscriptionality Mol cell rev 2009

Fast elongation favors exon skipping whereas slow elongation favors exon inclusion

De la mata

Elongation control in process coupling

What don’t we know about the CTD?

1. What is the role of Ser-7 phosphorylation? Ser-7 shows high phosphorylation across highly transcribed protein coding genes in S. cerevisae, but no role yet ascribed to this modification

2. What is the significance of different markings when comparing non-coding and protein coding genes and how is this difference set up?

3. To what extent do interdependent and co-occurrence of marks set-up bivalent/multivalent recognition patterns

See Tietjen…..Ansari NMSB(2010) 17: 1154Mayer….Cramer NMSB (2010): 1272

4. Genome wide ChIP analysis indicates some factors thought to be recruited by Ser-2 phosphorylation appear either signficantly prior to or after that event. Explanation?

NusG orthologue Spt5 functions with Spt4 ( and other proteins) in elongation control

Spt5: essential in yeast

A promoter proximal pause is characteristic of transcription of many genes in higher eukaryotes

Paused polymerase

Characteristics of paused polymerase ( pioneering work by John Lis Hsp70 locus in Drosophila)

1.In open complex ( KMnO4 footprinting)2.Some fraction can elongate (nuclear run-on experiments)

Later work:3. Ser-5 phosphorylated on CTD 4. Spt4/5 (DSIF) and NELF associated with paused polymerase ( ChIP; + required to recapitulate pause in vitro )

What triggers release to productive elongation?1)pTEFb phosphorylates Spt5 releasing it to move with RNA P; and a subunit of NELF, causing dissociation

2) Backtracking relieved ( SII)

Genome wide studies sequencing 5’ capped short mRNAs found them associated with ~30% of all genes in Drosophila; positions of their 3’ ends correspond to positions of stalled polymerase, and were also regions of high GC content; length of short mRNAs increases when SII is depleted suggesting that paused polymerases had backtracked and their mRNAs had been cleaved by SII

Adelman, Science, Cell 2010

NRG ( 2012) 13: 720

Potential roles of Paused Polymerase

Spt4/5 is also connected to other elongation complexes

Using activity based assay, Spt4/5, PAF and Tat-SF1 required for efficient elongation (DNA template)

Spt4/5PAF

Tat-SF1Physical interaction

Phosphorylation of Spt5 CTD by Bur-1 required for PAFentry into elongation complex

Using chromatin template and completely reconstituted factors, PAF stimulates elongation synergistically with TFIIS (independent of other activities of the PAF complex)

PAF

PAF TFIIS Physical interaction

∆PAF ∆TFIIS Synthetic lethal

Each elongation factor also interacts with RNAP

NusG may also mediate ribosome/ RNAP interaction

NusE is ribosomal protein S10, and structural studies indicate that its binding site would be exposed when S10 is part of the ribosome. This protein protein interaction could connect these two major macromolecular machines

CTD NusE50 µM

Altering translation rate alters the transcription rate

Condition translation rate transcription rate - 14 aa/sec 42 nt/sec+ chloramphenicol ( 1µg/ml) 9 aa/sec 27 nt/secSlow ribosome (streptomycin dependent) 6 aa/sec 19 nt/secSlow ribosome (+ streptomycin) 10 aa/sec 31 nt/sec

Footprinting studies show that the presence of a ribosome behind RNA polymerase prevents backtracking!

This could be a general mechanism to couple the rates of transcription and translation