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Biochem 201
Biological Regulatory Mechanisms
Lecture #2, Carol Gross; January 21, 2016
Regulation 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 9:973979.
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.
The dynamics of lac Repressor binding to its operator
Elf, 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 synthesis
Li, 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):99104. 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 elongation
Larson 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
molecules observed at near-base-pair resolution.
Nature. 2003 Dec 11;426(6967):684-7. Epub 2003 Nov 23.
Backtracking by single RNA polymerase
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
1. Quick review of protein-DNA interactions
2. Overview of bacterial gene regulation
3. Regulating transcription in the cellular milieu
4. An in-depth look at activation and repression
5. Regulatory circuits
6. Elongation control
How proteins recognize DNA
All 4 bp can be distinguished in the major groove
Common families of DNA
binding proteins
Overview: Every step of transcription can be regulated
NTPs
KB
R+P
RPc
initial
binding
Kf
Abortive
Initiation
RPo
Elongating
Complex
“isomerization”
DNA Binding Proteins used to alter promoter properties
Negative control: repressors prevent RNAP binding
R
-35
-10
Positive control: activators facilitate RNAP binding-favorable proteinprotein contact
Favorable
contact
A
*
RNAP holo
-35
-10
Gene regulation in E. coli: The Broad Perspective
• 3.6 mB chromosome
• 4400 genes
• 300-350 sequence-specific DNA-binding proteins
• 7  factors
In E. coli 1 copy/cell ≈ 10-9 M
If KD = 10-9M and things are simple:
10 copies/cell
100 copies/cell
occupied
90% occupied
99%
Construction of an effective activation system
Activating 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
Strategies to identify point of contact between activator and RNAP
1. Isolate “positive control” (pc)
mutations in activator. 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 activator-nonresponsive mutations in RNAP
S-S-X*
RNAP
-35
-10
M
RNAP
-35
-10
Construction of an effective repression system
Lac ~ 1980
-35
-10
Lac operator (O1)
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
Effects of looping (2 operators)
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.
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.
Allows control of gene regulation on multiple time scales through different
kinds of dissociation events
Partial dissociation: can initiate 1round of transcription (~10-20 molecules)
Full dissociation: 6 min to find site again
Regulatory Circuits are composed of network motifs
Negative feedback loops: tunes expression to cellular state
Blue line: negative feedback
Red line: constant rate of A synthesis unaffected by R
Positive feed back loops
Positive feedback loops can generate bistability and switch-like responses
Bistability at the lac operon
A P
O
lacZ
lacY
Permease
lacA
Repressor
(imports inducer)
Permease-YFP
Combinatorial control of gene expression
AND NOT Logic,
e.g. lac operon
AND Logic;
e.g. arabinose operon
AND NOT logic is used to regulate how E. coli responds to
sugar source
The CAP activator senses nutritional state
cAMP
Inactive CAP
high glucose
Active CAP—binds DNA
Regulates >100 genes positively or negatively
A P
O
lacZ
lacY
lacA
Repressor
Activator
CAP-cAMP
Activation of lac requires binding of the activator (high cAMP; no glucose)
AND NOT binding of the repressor (presence of lactose)
Coherent feed-forward loop allows timing of responses
Example: response to sugars
Transient input
CAP-cAMP
MalT activator
Sustained input
Regulated Elongation
Co-evolution of RNAP and Nascent RNA produced
functional interplay.
RNAP influence on RNA structure: vectorial transcription limits folding landscape
RNA influence on RNAP: sequence and structure alter polymerase speed up to 2
orders of magnitude, as well as functionality.
Current view of Pausing
(?)
Elemental Pause Elongation Complex
Formation of an RNA hairpin and its effect on RNAP
1.
Assemble complex on a synthetic scaffold
with a single fluorescent base (*)
2. Add antisense RNA such that RNA stem starts
12 bp from 3’ end of RNA; measure rate of
quenching. This mimics a hairpin pause; such
hairpins form inside the RNA exit channel
*
Result: rate of formation slows only 2-fold
3. Flap tip required to interact with duplex for pause
4. Clamp must be in open position for pause
Hein…& Landick Nature Structural & Molecular Biology 21, 794–802 (2014) doi:10.1038/nsmb.2867
Using a hairpin pause to couple transcription to translation
in operons encoding enzymes for biosynthesis of amino acids
1.
Pause RNA polymerase at a hairpin pause positioned immediately upstream of a terminator hairpin
2. Pausing of transcription allows initiation of translation; RNA polymerase releases from the pause when
the ribosome approaches thus allowing synchronized transcription/translation
3. The ribosome is translating a small ORF with several codons for the amino acid produced by the
biosynthetic genes in the operon e.g. hisitidine
4. If translation does not stall ( e.g. Amino acid in excess) the terminator hairpin is formed and very little of
the mRNA for the biosynthetic genes are produced.
5. If translation and the ribosome stall because amino acid is limiting, then the terminator loop does not form and
Abundant mRNA for the biosynthetic genes are formed.
6. This method of regulation is called Attenuation control
Attenuation in biosynthetic operons
His codons
TAA
hisL
1
hisG
2
No protein
synthesis
hisL
3
4
1 2
pause
hairpin
3 4
hisG
transcription
terminator
TAA
High
His
3 4
hisL
1
2
hisG
transcription
terminator
Operon mRNA
level
TAA
Low
His
hisL
2 3
1 transcription 4
anti-terminator
Low
hisG
High
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
Pauses can also be measured genome wide using NET-seq
Matt Larson ( Weissman lab)
RNA polymerase pausing is enriched at translation start site
Matthew H. Larson et al. Science 2014;344:1042-1047
Published by AAAS
E. coli NusG: A 21kD essential elongation factor
NTD
NGN domain
CTD
KOW domain
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 CTD of NusG interacts with other protein partners
NusG
CTD
50 µM
NusE
Rho
NusE, a ribosomal protein (S10) 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
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