Transcription and Its Regulation (Bioreg 2015 – Carol A. Gross)

January 20 –The Transcription CycleJanuary 22– Regulation of Transcription in ProkaryotesJanuary 26–Regulation of transcription in EukaryotesJanuary 29– In class discussion of problem set

Mechanism of Transcription Initiation

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.

2. ReviewsMurakami KS, Darst SA. (2003) Bacterial RNA polymerases: the wholo story. Curr Opin Struct Biol 13:31-9.

Campbell, E, Westblade, L, Darst, S., (2008) Regulation of bacterial RNA polymerase factor activity: a structural perspective. Current Opinion in Micro. 11:121-127

Herbert, KM, Greenleaf, WJ, Block, S. (2008) Single-Molecule studies of RNA polymerase: Motoring Along. Annu Rev Biochem. 77:149-76.

Werner, Finn and Dina Grohmann (201). Evolution of multisubunit RNA polymerases in the three domains of life. Nature Rev. Microbiology 9: 85-98

Grunberg, S. and Steven Hahn (2013) Structural Insights into transcription initiation by RNA polymerase II. TIBS 38: 603-11.

3. Studies of Transcription InitiationRoy S, Lim HM, Liu M, Adhya S. (2004) Asynchronous basepair openings in transcription initiation: CRP enhances the rate-limiting step. EMBO J. 23:869-75.

Sorenson MK, Darst SA. (2006).Disulfide cross-linking indicates that FlgM-bound and free sigma28 adopt similar conformations. Proc Natl Acad Sci U S A. 103:16722-7.

Young BA, Gruber TM, Gross CA. (2004) Minimal machinery of RNA polymerase holoenzyme sufficient for promoter melting. Science. 303:1382-1384

*Kapanidis, AN, Margeat, E, Ho, SO,.Ebright, RH. (2006) Initial transcription by RNA polymerase proceeds through a DNA-scrunching mechanism. Science. 314:1144-1147.

Revyakin A, Liu C, Ebright RH, Strick TR (2006) Abortive initiation and productive initiation by RNA polymerase involve DNA scrunching. Science. 314: 1139-43.

Murakami KS, Masuda S, Campbell EA, Muzzin O, Darst SA (2002). Structural basis of transcription initiation: an RNA polymerase holoenzyme-DNA complex. Science. 296:1285-90.

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.

Error correction

*Vassylyev, DG, Vassylyeva, MN, Zhang, J, Landick, R (2007). Structural basis for substrate loading in bacterial RNA polymerase. Nature, 448(7150), 163-8.

*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.

Discussion Paper**Feklistov A and Darst, SA (2011) Structural basis for Promoter -10 Element recognition by the Bacterial RNA Polymerase s Subunit. Cell 147: 1257 – 1269Accompanying preview: Liu X, Bushnell DA and Kornberg RD ( 2011) Lock and Key to Transcription:s –DNA Interaction. Cell: 147: 1218-1219

*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.

Key Points 1. Multisubunit RNA polymerases are conserved among all organisms

2. RNA polymerases cannot initiate transcription on their own. In bacteria s70 is required to initiate

transcription at most promoters. Among other functions, it recognizes the key features of most bacterial promoters, the -10 and -35 sequences.

2. E. coli RNA polymerase holoenzyme, (core + s) finds promoter sequences by sliding along DNA and by transfer from one DNA segment to another. This behavior greatly speeds up the search for specific DNA sequences in the cell and probably applies to all sequence-specific DNA-binding proteins.

3. Transcription initiation proceeds through a series of structural changes in RNA polymerase, s70 and DNA.

4. A key intermediate in E. coli transcription initiation is the open complex, in which the RNA polymerase holoenzyme is bound at the promoter and ~12 bp of DNA are unwound at the transcription startpoint. Open complex formation does not require nucleoside triphosphates. Its presence can be monitored by a variety of biochemical and structural techniques.

5. Recognition of the -10 element of the promoter DNA is coupled with strand separation

6. When the open complex is given NTPs, it begins the ‘abortive initiation’ phase, in which RNA chains of 5-10 nucleotides are continually synthesized and released.

7. Through a “DNA scrunching” mechanism the energy captured during synthesis of one of these short

transcripts eventually breaks the enzyme loose from its tight connection to the promoter DNA, and it begins

the elongation phase.

8. Aspects of the mechanism of initiation are likely to be conserved in eukaryotic RNA polymerase

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.

rRNAs snRNAs miRNAs

Other non-coding RNAs (e.g. telomerase RNA)

mRNAs

translation

proteins

transcription

(RNA processing)

Transcription is Important

rRNAs snRNAs miRNAs

Other non-coding RNAs (e.g. telomerase RNA)

mRNAs

translation

proteins

Transcription is Important

transcription

RNA processing

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

KB Kf

initial binding

“isomerization”

Abortive Initiation

ElongatingComplex RPoRPcR+P

NTPs

Steps in transcription

Initiation transition Elongation/termination

Structure of RNAP in the three domains

Werner and Grohmann (2011),Nature Rev Micro 9:85-98

Extra RNAP subunits provide interaction sites for transcription factors, DNA and RNA, and modulate diverse RNAP activities

Bacteria

Universally conservedArchaeal/eukaryotic

Archaea Eukarya

Transcription

Initiation of transcription presents challenges to the cell

1. RNAP is specialized to ELONGATE, not INITIATE

2. Initiating RNAP must open DNA to permit transcription

‘holoenzyme’

'

KD ~ 10-9 M

+

‘core’}

Can begin transcription on

promoters and can elongate

}Can elongate but

cannot begin transcription at

promoters

factor is required for bacterial RNA polymerase to initiate transcription on promoters

'

The discovery of initiation factors

How was discovered (Burgess, 1969)

Identification of cellular RNA polymerase

E.coli lysate

buffer

*ATPCTPGTPUTP

Calf thymus DNA

Look for incorporation of *ATP into RNA chains

Initial purification ofRNA polymerase

Lysate

various fractionation steps (DEAE column, glycerol gradient etc)

Active fractions identified by assay

Labmate Jeff Roberts reported that the new, improved preparation of RNAP (peak 2) had no activity on DNA

Peak 1 restored activity

Improved purification of RNA polymerase leads to the discovery of s

Improved fractionationlysate

phosphocellulose column

salt

OD

28

0

1

2

Act

ivit

y (

*ATP)

CT D

NA

Fraction #

SDS gel analysis Peak 1 Peak 2

'

increases rate of initiation

g

Transc

ripti

on

D

NA Assay:

incorporation P ATP using l as template

Is the -10 promoter element recognized as Duplex or SS DNA?

-10 logo-35 logo

Helix-turn-helix in Domain 4Recognizes -35 as duplex DNA

Recognition of the Prokaryotic promoter

s is positioned for DNA recognition

Transition to elongation: Abortive initiation

KB Kf

initial binding

“isomerization”

Abortive Initiation

ElongatingComplex RPoRPcR+P

NTPs

Abortive Initiation and Promoter escape

During abortive initiation, RNAP synthesizes many short transcripts, but reinitiates rapidly.

How can the active site of RNAP move forward along the DNA while maintaining

contact with the promoter?

Three models for Abortive initiation

#1

Predicts expansion and contraction of RNAP

Predicts expansion and contraction of DNA

Predicts movement of both the RNAP leading and trailing edge relative to DNA

#2

#3

Förster (fluorescence) resonance energy transfer (FRET) allows the determination of intramolecular distances through fluorescent coupling between a donor (yellow star) and an acceptor (red star) dye. When the donor (yellow star) is excited (blue arrows) it emits light. When the donor fluorophore moves sufficiently close to the acceptor (right), resonance energy transfer results in emission of a longer wavelength by the acceptor. The degree of acceptor emission relative to donor excitation is sensitive to the distance between the attached dyes.This process depends on the inverse sixth power of the distance between fluorophores. By measuring the intensity change in acceptor fluorescence, distances on the order of nanometers can currently be measured in single molecules with millisecond time resolution

Experimental set-up for single molecule FRET: Single transcription complexes labeled with a fluorescent donor (D, green) and a fluorescent acceptor (A, red) are illuminated as they diffuse through a femtoliter-scale observation volume (green oval; transit time ~1 ms); observed in confocal microscope

Using single molecule FRET to monitor movement of RNAP and DNA

A. N. Kapanidis et al., Science 314, 1144 -1147 (2006)

Initial transcription involves DNA scrunching

Lower E* peak is free DNA; higher E* peak is DNA in open complex; distance is shorter because RNAP

induces DNA bending

Open complex

Initial transcription involves DNA scrunching

Higher E* in Abortive initiation complex than open complex results from DNA scrunching

Open complex

Abortive initiation complex

Initial transcription involves DNA scrunching

Open complex

Abortive initiation complex

At a typical promoter, promoter escape occurs only after synthesis of an RNA product ~9 to 11 nt in length (1–11) and thus can be inferred to require scrunching of ~7 to 9 bp (N – 2, where N = ~9 to 11; Fig. 3C). Assuming an energetic cost of base-pair breakage of ~2 kcal/mol per bp (30), it can be inferred that, at a typical promoter, a total of ~14 to 18 kcal/mol of base-pair–breakage energy is accumulated in the stressed intermediate. This free energy is high relative to the free energies for RNAP-promoter interaction [~7 to 9 kcal/mol for sequence-specific component of RNAP-promoter interaction (1)] and RNAP-initiation-factor interaction [~13 kcal/mol for transcription initiation factor {sigma}70 (31)].

The energy accumulated in the DNA scrunched “stressed intermediate could disrupt interactions between RNAP,

and the promoter, thereby driving the transition from initiation to elongation

s is positioned to block elongating transcripts

Elongation

Steps in the Nucleotide Addition Cycle ( NAC)

NTP binding

Nucleotide addition:Pretranslocated state

Post-translocated state

Backtracked state

NTP a

dd

itio

n

rate

lim

itin

g

Cutaway view of elongating complex

Structure of RNAP

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)

Mg++

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

How were they able to get a structure given than RNA polymerase backtracks

Complexes were used to determine RNAP structure during nucleotide addition

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

The Transcript Cleavage Reaction

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

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

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

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)

Current view of Pausing

(?)

Elemental Pause Elongation Complex

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

Cellular RNA polymerases in all living organisms are evolutionary related

LUCA-Last universal common ancestor

s

Gre

LUCA may have had elongating, not initiating RNA polymerase