Control of Transcription Initiation

General References

Chapter 16 of Molecular Biology of the Gene 6th Edition (2008) by Watson, JD, Baker, TA, Bell, SP, Gann, A, Levine, M, Losick, R. 547-587.

Ptashne, M. and Gann, A. (2002) Genes and Signals. Cold Spring Harbor Laboratory Press, Cold Spring Harbor.

Luscombe, N.M., Austin, S.E., Berman, H.M., Thornton, J.M. (2000) An overview of the structures of protein-DNA complexes. Genome Biology 1(1): reviews001.1-001.37

Examples of Control Mechanisms

Alternative Sigma Factors

Sorenson, MK, Ray, SS, Darst, SA (2004) Crystal structure of the flagellar sigma/anti-sigma complex 28 /FlgM reveals an intact sigma factor in an inactive conformation. Molecular Cell 14:127-138.Gruber, TM, Gross, CA (2003) Multiple sigma subunits and the partitioning of bacterial transcription space. Annu. Rev. Microbiol 57:441-66

Increasing the Initial Binding of RNA Polymerase Holoenzyme to DNA

Lawson CL, Swigon D, Murakami KS, Darst SA, Berman HM, Ebright RH. (2004) Catabolite activator protein: DNA binding and transcription activation. Curr Opin Struct Biol. 14:10-20.

Increasing the Rate of Isomerization of RNA Polymerase

*Dove, S.L., Huang, F.W., and Hochschild, A. (2000) Mechanism for a transcriptional activator that works at the isomerization step. Proc Natl Acad Sci USA 97: 13215-13220.Jain, D. Nickels, B.E., Sun, L., Hochschild, A., and Darst, S.A. (2004) Structure of a ternary transcription activation complex. Mol Cell 13: 45-53.Hawley and McClure (1982) Mechanism of Activation of Transcription from the PRM promoter. JMB 157: 493-525

DNA looping**Oehler, S., Eismann, E.R., Kramer, H. and Mueller-Hill, B. (1990) The three operators of the lac operon cooperate in repression. EMBO 9:973-979.Vilar, J.M.G. and Leibler, S. (2003) DNA looping and physical constraints on transcription regulation. J Mol Biol 331:981-989.

Dodd, I.B., Shearwin, K.E., Perkins, A.J., Burr, T., Hochschild, A., and Egan, J.B. (2004) Cooperativity in long-range gene regulation by the cI repressor. Genes Dev. 18:344-354.

The dynamics of lac Repressor binding to its operatorElf, J., Li, G.W., and Xie, X.S. (2007). Probing transcription factor dynamics at the single-molecule level in a living cell. Science 316, 1191–1194. Li, G.W., Berg, O.G., and Elf, J. (2009). Effects of macromolecular crowding and DNA looping on gene regulation kinetics. Nat. Phys. 5, 294–297 Li, G.W., and Xie, X.S. (2011). Central dogma at the single-molecule level in living cells. Nature 475, 308–315. Hammar, P., Leroy, P., Mahmutovic, A., Marklund, E.G., Berg, O.G., and Elf, J. (2012). The lac repressor displays facilitated diffusion in living cells. Science 336, 1595–1598

*Choi, PJ, Cai,L, Frieda K and X. Sunney Xie (2008) A Stochastic Single-Molecule Event Triggers Phenotype Switching of a Bacterial Cell Science 2008: 442-446. [DOI:10.1126/science.1161427]

In vivo logic of absolute rates of protein synthesisLi, GW, Burkhardt D, Gross, C and Weissman JS (2014). Quantifying absolute protein synthesis rates reveals principles underlying allocation of cellular resources. Cell.157(3):624-35. doi: 10.1016

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.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. 2013 Jan 31;152(3):431-41. doi: 10.1016/j.cell.2012.12.020.

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

Measurement of elongationLarson MH, Mooney RA, Peters JM, Windgassen T, Nayak D, Gross CA, Block SM, Greenleaf WJ, Landick R, Weissman JS. Science. 2014: A pause sequence enriched at translation start sites drives transcription dynamics in vivo. May 30;344(6187):1042-7.

Shaevitz JW, Abbondanzieri EA, Landick R, Block SM Backtracking by single RNA polymerase molecules observed at near-base-pair resolution. Nature. 2003 Dec 11;426(6967):684-7. Epub 2003 Nov 23.

Important Points

1. Every step in transcription initiation can be regulated to increase or decrease the number of successful initiations per time.

2. In E. coli, transcription initiation is controlled primarily by alternative factors and by a large variety of other sequence-specific DNA-binding proteins.

3. G=RTlnKD. This means that a net increase of 1.4 kcal/mole (the approximate contribution of an additional hydrogen bond) increases binding affinity by 10-fold. Many examples of transcription activation in bacteria take advantage of such weak interactions.

4. To activate transcription at a given promoter by increasing KB, the concentration of RNA polymerase in the cell and its affinity for the promoter must be in the range so an increase in KB makes a difference. Likewise, to activate transcription by increasing kf, the rate of isomerization must be slow enough so the increase makes a substantial difference.

5. Network motifs give the regulatory circuit its properties

6. Transcriptional pauses are integral to the transcription process and are extensively utilized for regulatory roles

Transcriptional Control: Bacterial Paradigms

Every step of transcription can be regulated

KB Kf

initial binding

“isomerization”

Abortive Initiation

ElongatingComplex RPoRPcR+P

NTPs

DNA Binding Proteins used to alter promoter properties

How proteins recognize DNA

All 4 bp can be distinguished in the major groove

Common families of DNA binding proteins

Gene regulation in E. coli: The Broad Perspective

• 4400 genes

• 300-350 sequence-specific DNA-binding proteins

• 7 factors

In E. coli 1 copy/cell ≈ 10-9 MIf KD = 10-9M and things are simple:

10 copies/cell 90% occupied100 copies/cell 99% occupied

Regulation by repressors and activators

Case Study: How bacteria monitor and respond to nutrient status

Regulation of the lactose utilization operon: Dual negative and positive control

O lacZ lacY lacAPA

Repressor

ActivatorCAP-cAMP

KB Kf

initial binding

“isomerization”

Abortive Initiation RPoRPcR+P

NTPs

Lac repressor and DNA looping

-35 -10

Lac operator

Lac ~ 1980

What is the function of these weak operators?

O2 1/10 affinity of O1

O3 1/300 affinity of O1

Lac 2000-35 -10-90

O3 O1 O2

+400

Oehler, 2000

The weak operators significantly enhance represssion

Oehler, 2000

OKOm

Through DNA looping, Lac repressor binding to a “strong” operator (Om) can be helped by binding to a “weak” operator (OA)

Om

Oa

Better!

M MA mutant Lac repressor that cannot formtetramers is not helped by a weak site

Om (main operator) binds repressor more tightly than Oa (auxiliary operator). Transcription takes place only in the states (i) and (iii), when Om is not occupied.

Effects of looping (2 operators)

Allows control of gene regulation on multiple time scales through different kinds of dissociation events

Vilar, J.M.G. and Leibler, S. (2003) J Mol Biol 331:981-989

One operator: a single unbinding event is enough for the repressor to completely leave the neighborhood of the main operator.

Two operators: repressor can escape the neighborhood of the main operator only if it sequentially unbinds both operators.

Partial dissociation: can initiate 1round of transcription (~10-20 molecules)

Full dissociation: 6 min to find site again; allows establishing bistability

I. Activating transcription initiation at KB (initial binding) step

∆ G = RT lnKD; if * nets 1.4 kcal/mol, KB goes up 10-fold

Positive control: activators ( e.g. CAP); facilitate RNAP binding with favorable protein-protein contact

A

-35 -10

RNAP holoFavorable contact

*

Activating by increasing KB is effective only if initial promoter occupancy is low

If favorable contact nets 1.4Kcal/mole (KB goes up 10X) then:

Transcription rate increases 10-fold

Little or no effect on transcription rate

RNAP

99% occupied

A RNAP

99.9% occupied

*

b) If initial occupancy of promoter is high

a) If initial occupancy of promoter is low

1% occupied

RNAP

10% occupied

A RNAP*

A case study of activation at KB: CAP at the lac operon:

CAP increases transcription ~40-fold; KB ; no effect on kf

CAP at lac operon

Inactive CAP Active CAP

Regulates >100 genes positively or negatively

cAMP high glucose

How is CAP activated?

Strategies to identify point of contact between CAP and RNAP

1. Isolate “positive control” (pc) mutations in CAP. These mutant proteins bind DNA normally but do not activate transcription

MM

3. Isolate CAP-non-responsive mutations in -CTD

-35 -10

M

RNAP

2. “Label transfer” (in vitro) from activator labeled near putative “pc” site to RNAP

Activate X*; reduce S-S; X* is transferred to nearest site; determine location by protein cleavage studies; X* transferred to -CTD

-35 -10

S-S-X*

RNAP

Summary: Stereotypical binding of repressors and activators regulates transcription initiation

Negative control: repressors (e.g. , Lac ); prevent RNAP binding

R

-35 -10Positive control: activators ( e.g. CAP); facilitate RNAP binding with favorable protein-protein contact

A

-35 -10

RNAP holoFavorable contact

*

KB Kf

initial binding

“isomerization”

Abortive Initiation

ElongatingComplex RPoRPcR+P

NTPs

Regulatory Circuits are composed of network motifs

Negative feedback loops: tunes expression to cellular state

Blue line: negative feedbackRed line: constant rate of A synthesis unaffected by R

Positive feed back loops can generate bistability

Combinatorial control of gene expression

AND NOT Logic, e.g. lac operon

AND Logic;e.g. arabinose operon

Regulated 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

Stall(3 NTP’s)

Start reaction with 4th *NTP+ heparin to prevent reinitiation

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, one with long pause

*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)

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

Pauses can also be measured genome wide using NET-seq

Matt Larson ( Weissman lab)

Current view of Pausing

(?)

Elemental Pause Elongation Complex

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

hisL hisG

TAA

1 2 3 4

hisL hisG

TAA

3 4High His

1 2transcriptionterminator

1 4

hisL hisG

TAA

2 3Low His

transcriptionanti-terminator

High

Operon mRNAlevel

Low

Attenuation in biosynthetic operonsHis codons

hisL hisG3 4No proteinsynthesis

transcriptionterminator

1 2

pausehairpin

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

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

The CTD of NusG interacts with other protein partners

NusGCTD

NusE, a ribosomal protein (S10) 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

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

regulators