Biochemistry 201Biological Regulatory Mechanisms: Lecture 3January 28, 2013

Control of Transcription in Bacteria

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.

*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]

Attenuation/riboswitchesMerino E and Yanofsky, C. (2005) Transcription attenuation: A highly conserved regulatory strategy used by bacteria. Trends in Genetics 21: 260 - 262

Winkler WC, Breaker RR. (2005) Regulation of bacterial gene expression by riboswitches. Annu Rev Microbiol 59:487-517.Landick R. (2009) Transcriptional pausing without backtracking. Proc Natl Acad Sci 106:8797-8.

Serganov a and E. Nudler (2012) A decade of riboswitches. Cell 152: 17-24 (Review)

Xia et al (2012): Riboswitch Control of Aminoglycoside Antibiotic resistance. Cell 152: 68 - 81

NusG and General Elongation ControlMooney, R………and Landick R. ( 2010) Two Structurally Independent Domains of E. coli NusG Create Regulatory Plasticity via Distinct Interactions with RNA Polymerase and Regulators. JMB 391: 341-351

Herbert, KM……Landick, R and Block, S. (2010) E. coli NusG Inhibits Backtracking and Accelerates Pause-Free Transcription by Promoting Forward Translocation of RNA Polymerase. JMB 399: 17 -30

Klein, B.,….and Murakami K. ( 2011). RNA polymerase and transcription elongation factor Spt4/5 complex structure. PNAS 108: 546-50

Coupling of translation and transcriptionBurmann, B…..Gottesman, M and Rosch, P. ( 2010) A NusE:NusG Complex Links Transcription and Translation Science 328: 501-4

*Proshkin, S..and Nudler, E. (2010). Cooperation Between Translating Ribosomes and RNA Polymerase in Transcription Elongation. Science 328: 504 -8

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 k f, the rate of isomerization must be slow enough so the increase makes a substantial difference.

5. DNA looping allows proteins bound to distant sites on DNA to interact.

6. Transcriptional pausing and alternative RNA structures underlie many elongation control mechanisms.

Control of Transcription in Bacteria

Every step of transcription can be regulated

KB Kf

initial binding

“isomerization”

Abortive Initiation

ElongatingComplex RPoRPcR+P

NTPs

Gene regulation in E. coli: The Broad Perspective

• 4400 genes

• 300-350 sequence-specific DNA-binding proteins

• 7 factors

Alternative s are major control mechanism in bacteria

The Number of Sigma Factors Varies Dramatically among Bacteria

Mycoplasma sp. 1Aquifex aeolicus 4Escherichia coli 7Bacillus subtilis 18Pseudomonas aeruginosa 24Streptomyces coelicolor 63

Alternative s

Alternative s direct RNAP to a discrete promoter set in response to a specific condition

Figure 7–63 Interchangeable RNA polymerase subunits as a strategy to control gene expression in a bacterial virus. The bacterial virus SPO1, which infects the bacterium B. subtilis, uses the bacterial polymerase to transcribe its early genes immediately after the viral DNA enters the cell. One of the early genes, called 28, encodes a sigmalike factor that binds to RNA polymerase and displaces the bacterial sigma factor. This new form of polymerase specifically initiates transcription of the SPO1 “middle” genes. One of the middle genes encodes a second sigmalike factor, 34, that displaces the 28 product and directs RNA polymerase to transcribe the “late” genes.This last set of genes produces the proteins that package the virus chromosome into a virus coat and lyse the cell. By this strategy, sets of virus genes are expressed in the order in which they are needed; this ensures a rapid and efficient viral replication. From Molecular Biology of the Cell, 4th Edition.

Tremendous Diversity Among the Minimal Sigma Class

Regulation by repressors and activators(alter reactivity of 70-holoenzyme)

A brief digression: How proteins recognize DNA

All 4 bp can be distinguished in the major groove

In vivo parameters for Sequence-Specific DNA binding proteins

KD ≈ 10-6 - 10-10M in vivo In E. coli 1 copy/cell ≈ 10-9 M

If KD = 10-9M and things are simple: 10 copies/cell 90% occupied 100 copies/cell 99% occupied

Common families of DNA binding proteins

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

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

R

-35 -10

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

A

-35 -10

RNAP holoFavorable contact

*

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

Representative states of the binding of the repressor to one operator (top panel) or to two operators (bottom panel). 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. The arrows indicate the possible transitions between states. Note that with one operator, a single unbinding event is enough for the repressor to completely leave the neighborhood of the main operator. With two operators, the repressor can escape from the neighborhood of the main operator only if it unbinds sequentially both operators.

From: Vilar, J.M.G. and Leibler, S. (2003) DNA looping and physical constraints on transcription regulation. J Mol Biol 331:981-989

.

Theoretical consideration of effects of looping (2 operators)

I. Regulating 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

II. Regulating transcription initiation at kf (isomerization) step

PRM 107 M-1 7 X 10-4/sec 16 min

PRM + C1 at OR2 107 M-1 7 X 10-3/sec 1.6 min

KB kf 1/2 time O.C. formation

___________________________________________________________________________________________________________________________________________

“isomerization”

KB Kf

initial binding

Abortive Initiation

ElongatingComplex RPoRPcR+P

λcI binds cooperatively to operator sites OR1 and OR2 and interacts with to activate transcription from PRM

Case study: repressor at PRM

The interactions between cI and are well established

c) In an artificial construct, cI “recruits” Domain 4 to the promoter

d) Co-crystal of cI and Domain 4 on promoter reveals expected contacts and noconformational changes

Why then does cI function at kf (post-recruitment) not at KB?

b) “bypass mutants in Domain 4

Mutagenize rpoD plasmid

Introduce into E. coli

Isolate mutants that restore activation by pc Asp38 Asn38

rpo D

Arg596 His596

a) “pc” mutants in cI

Model for mechanism of action of λcI at PRM

Dove S L et al. PNAS 2000;97:13215-13220

In the absence of cI, formation of an unproductive intermediate limits open complex formation at PRM

Activating region and its target (red patches) are misaligned in the closed complex but come into alignment subsequently during the process of open complex formation. Depicted in brackets is a hypothetical productive intermediate that is stabilized by λ cI.

Attenuation control

Promoting either elongation or termination by stabilizing alternative 2˚structures of mRNA

Case study: ”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

General elongation control: NusG

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

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

Case study: role of NusG: An 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?

The NTD of NusG is sufficient to enhance elongation rate

and to prevent backtracking!

The NusG NTD interacts with RNAP (coiled coiled motif in ’)

Current view of Pausing

(?)

Elemental Pause Elongation Complex

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

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

Coupled syntheses.

J W Roberts Science 2010;328:436-437

Published by AAAS

Single molecule experiments indicate that NusG suppressesbacktracking, decreases the frequency of “elemental pause”, and modestly increases the pause-free elongation rate

A unified pathway for elongation and pausing. The main pathway for transcript elongation is shown (light blue boxes; top row; adapted from Ref. 38). In a Brownian ratchet mechanism, RNAP oscillates stochastically between pre- and post-translocated states prior to the reversible binding of NTP followed by the (nearly) irreversible condensation reaction and pyrophosphate release, which rectify this motion in the transcriptionally downstream direction. The displacement associated with translocation, δ, corresponds to the longitudinal distance subtended by a single base pair. The elemental pause state is depicted (middle row, orange box; adapted from Ref. 14), shown branching from the pre-translocated state: entry into this state does not involve translocation. The long-lifetime, backtracked pause state (bottom row; orange box) is entered via the elemental pause state and involves the upstream translocation of RNAP through one or more base pairs, Nδ. Our modeling suggests that the addition of NusG promotes the downstream motion of RNAP, affecting those transitions that involve translocation (red arrows).