Biochemistry 201
Biological Regulatory Mechanisms: Lecture 3
January 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 l 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 l 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/riboswitches
Merino 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 Control
Mooney, 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 transcription
Burmann, 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 kf, 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
NTPs
KB
R+P
RPc
initial
binding
Kf
RPo
“isomerization”
Abortive
Initiation
Elongating
Complex
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
Alternative s
The Number of Sigma Factors Varies Dramatically among Bacteria
Mycoplasma sp.
Aquifex aeolicus
Escherichia coli
Bacillus subtilis
Pseudomonas aeruginosa
Streptomyces coelicolor
1
4
7
18
24
63
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
Common families of DNA
binding proteins
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
I. Regulating transcription initiation at KB (initial binding) step
Negative control: repressors (e.g. l, Lac ); prevent RNAP binding
R
-35
-10
Positive control: activators ( e.g. CAP); facilitate RNAP binding with
favorable protein-protein contact
Favorable
contact
A
*
RNAP holo
-35
-10
Lac repressor and DNA looping
Lac ~ 1980
-35
-10
Lac operator
Lac 2000
O3
-90
O1
-35
-10
O2
+400
Oehler, 2000
O2
1/10
affinity of O1
O3
1/300 affinity of O1
What is the function of these weak operators?
The weak operators significantly enhance represssion
Oehler, 2000
Through DNA looping, Lac repressor binding to a “strong” operator (Om)
can be helped by binding to a “weak” operator (OA)
OK
Om
Oa
Better!
Om
A mutant Lac repressor that cannot form
tetramers is not helped by a weak site
MM
Theoretical consideration of effects of looping (2 operators)
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
.
I. Regulating transcription initiation at KB (initial binding) step
Positive control: activators ( e.g. CAP); facilitate RNAP binding with
favorable protein-protein contact
Favorable
contact
A
*
RNAP holo
-35
-10
∆ G = RT lnKD; if * nets 1.4 kcal/mol, KB goes up 10-fold
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:
a) If initial occupancy of promoter is low
RNAP
A *
RNAP
10% occupied
1% occupied
Transcription rate increases 10-fold
b) If initial occupancy of promoter is high
RNAP
99% occupied
A *
RNAP
99.9% occupied
Little or no effect on transcription rate
A case study of activation at KB: CAP at the lac operon:
How is CAP activated?
cAMP
Inactive CAP
high glucose
Active CAP
Regulates >100 genes positively or negatively
CAP at lac operon
CAP increases transcription ~40-fold; KB ; no effect on kf
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
M
M
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
3. Isolate CAP-non-responsive
mutations in -CTD
S-S-X*
RNAP
-35
-10
M
RNAP
-35
-10
II. Regulating transcription initiation at kf (isomerization) step
KB
R+P
initial
binding
RPc
Kf
RPo
Abortive
Initiation
Elongating
Complex
“isomerization”
Case study: l repressor at PRM
λcI binds cooperatively to operator sites OR1 and OR2 and interacts with  to activate
transcription from PRM
KB
kf
1/2 time O.C. formation
__________________________________________________________________________________________________________________________________________
PRM
PRM + lC1 at OR2
_
107 M-1
7 X 10-4/sec
16 min
107 M-1
7 X 10-3/sec
1.6 min
The interactions between lcI and  are well established
a) “pc” mutants in lcI
b) “bypass mutants in  Domain 4

rpo D
Mutagenize rpoD plasmid
Introduce into E. coli
c) In an artificial construct, lcI “recruits”
 Domain 4 to the promoter
d) Co-crystal of lcI and  Domain 4 on
promoter reveals expected contacts and no
conformational changes
Isolate mutants that restore
activation by lpc Asp38 Asn38
 Arg596
Why then does lcI function at kf
His596
(post-recruitment)
not at KB?
Model for mechanism of action of λcI 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.
In the absence of lcI, formation of an unproductive intermediate limits open complex formation at PRM
Dove S L et al. PNAS 2000;97:13215-13220
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
1:2 is a pause hairpin
3:4 is an intrinsic terminator
Leader peptide has tryptophan residues
2:3 is an “antiterminator”
hairpin
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
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 b’)
Current view of Pausing
(?)
Elemental Pause Elongation Complex
The CTD of NusG interacts with other protein partners
CTD
50 µM
NusE
Rho
NusE is part of a complex of proteins mediating
antitermination/termination depending on its protein partners
Rho 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
CTD
50 µM
NusE
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
Altering translation rate alters the transcription rate
Condition
+ chloramphenicol ( 1µg/ml)
Slow ribosome (streptomycin dependent)
Slow ribosome (+ streptomycin)
translation rate
14 aa/sec
9 aa/sec
6 aa/sec
10 aa/sec
transcription rate
42 nt/sec
27 nt/sec
19 nt/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 suppresses
backtracking, 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).