Transcript Bioreg2017_Transcription1_Bacteriax

Transcription, chromatin and Its Regulation (Carol A. Gross; Geeta Narlikar)
January 19, 2017 – Transcription Initiation and its Regulation in Bacteria
January 23, 2017 – Transcription Initiation and its Regulation in Eukaryotes
January 26, 2016 – Chromatin 1
January 30, 2016 – Chromatin 2
February 2, 2016 – Transcription Elongation and its regulation in Bacteria and Eukaryotes
February 6, 2016 – In class discussion of problem set
Transcription Initiation and its Regulation in Bacteria
References
1. General
Chapter 12,16 of Molecular Biology of the Gene 6th Edition (2008) by Watson, JD, Baker, TA, Bell, SP, Gann, A, Levine, M,
Losick, R. 377-414.
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
2. Reviews
Murakami 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
perspective. Current Opinion in Micro. 11:121-127
factor activity: a structural
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: 60311.
3. Studies of Transcription Initiation
Roy S, Lim HM, Liu M, Adhya S. (2004) Asynchronous basepair openings in transcription initiation: CRP enhances the ratelimiting 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.
*Kapanidis, AN, Margeat, E, Ho, SO,.Ebright, RH. (2006) Initial transcription by RNA polymerase proceeds through a DNAscrunching 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.
4. 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.
5. 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 – 1269
Accompanying preview: Liu X, Bushnell DA and Kornberg RD ( 2011) Lock and Key to Transcription:
s –DNA Interaction. Cell: 147: 1218-1219
6. Examples of Control Mechanisms
a. Alternative Sigma Factors
Sorenson, MK, Ray, SS, Darst, SA (2004) Crystal structure of the flagellar sigma/anti-sigma complex s28 /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
b.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.
c.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
d. 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.
e. 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.
e. 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 322: 442-446. [DOI:10.1126/science.1161427]
f. 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
Important concepts
1. Cellular RNA polymerases are conserved across all organisms. These important machines not only produce the transcript but also play
regulatory roles.
2. The discrete requirements of initiation and elongation mean that all RNA polymerases have initiation subunits. In bacteria, sigma (s) is the
Initiation subunit.
3. The core prokaryotic promoter has two binding sites for s ( -35 and -10 nucleotides from the transcription start site). During initiation the
transcription start site is opened. Strand opening event initiates in the -10 region of the promoter.
4. Bacteria contain a single housekeeping s and multiple alternative s s, which generally coordinate responses to stress.
5. Transcription is regulated positively by activators and negatively by repressors. There are many quantitative considerations in designing
successful regulatory regimes. In particular, binding sites of RNA polymerase promoters) activators and repressors must be weak to achieve
meaningful regulation. Thus, these sites often differ significantly from the “consensus” binding sites that have been determined.
6. Bacterial activators and regulators bind very close to the promoter. Almost all activators directly contact RNA polymerase at either the
s or a subunit.
7. Regulatory circuits contain common network motifs. Negative and positive feedback loops are predominant motifs.
8. Regulatory circuits often combine motifs to achieve the desired response to an environmental state.
Outline
1. Introduction to Transcription/RNA polymerase
2. Bacterial paradigm for transcription initiation
A. Process of Transcription Initiation
B. Transition to elongation: Abortive Initiation
C. Regulating Transcription initiation
The Transcription cycle:
Initiation, Elongation, Termination
Binding: closed complex
Promoter melting: open complex
Initial transcribing complex
Elongation
Termination
A Schematic view of RNA polymerase
transcribing DNA
RNA polymerase (pale blue) moves stepwise along DNA unwinding the DNA at its active site indicated
by the Mg2+ (red), which is required for catalysis. The polymerase adds nucleotides to the RNA chain,
using the DNA in the active site as a template. The RNA/DNA hybrid is about 9 nt in length, after
which the RNA peels off and exits through the RNA exit channel. NTPs enter through the uptake
(secondary) channel. (adapted from MBOC p.304)
Structure of RNAP in the three domains
Universally conserved
Archaeal/eukaryotic
Bacteria
Archaea
Eukarya
Transcription
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
Cellular RNA polymerases are Important
1. Produce all RNAs in the cell at appropriate amount
2. Coordinate transcription in response to
environmental/developmental changes
3. Coordinate transcription with downstream events
Transcription initiation
Steps in Transcription Initiation
NTPs
KB
R+P
RPc
Kf
RPo
initial “isomerization”
binding
Initiation
Abortive
Initiation
transition
Elongating
Complex
Elongation/
termination
All cellular RNAPs have initiation subunits
The Bacterial paradigm for Initiation
Core RNAP + sigma
a2
s
Initiation factor
Holoenzyme
a2s
Improved purification of RNA polymerase leads to the discovery of s
lysate
Improved fractionation
OD 280
salt
2
1
Activity (*ATP)
CT DNA
phosphocellulose column
Fraction #
Peak 1
Peak 2
'
Peak 1 restored activity
increases rate of initiation
Transcription
DNA
SDS gel analysis
Labmate Jeff Roberts
reported that the new,
improved preparation of
RNAP (peak 2) had no
activity on l DNA
Assay:
incorporation 2P ATP
using l as template
s
Recognition of the Prokaryotic promoter
-35 logo
-10 logo
Helix-turn-helix in Domain 4
Recognizes -35 as duplex DNA
Is the -10 promoter element recognized as Duplex or SS DNA?
s is positioned for DNA recognition
Transition to elongation:
Abortive initiation
Abortive Initiation and Promoter escape
NTPs
KB
R+P
RPc
Kf
RPo
initial “isomerization”
binding
Abortive
Initiation
Elongating
Complex
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 movement of both the RNAP leading and trailing edge relative to DNA
#2
Predicts expansion and contraction of RNAP
#3
Predicts expansion and contraction of DNA
Science (2006 314:
1139-43; 1144-47;
Slide 38-41
Using single molecule FRET to monitor movement of RNAP and DNA
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
Conclusion: DNA shortens (scrunching!)
s is positioned to block elongating transcripts
In vitro transcription: #1 full-length s; #2: truncated s: no domain 4 or s-4 in exit tunnel) Murakami, Darst
2002
The Bacterial paradigm for Regulating
Initiation
Gene regulation in E. coli: The Broad Perspective
• 3.6 mB chromosome
• 4400 genes
• 7 s factors (housekeeping s and alternative ss)
• 300-350 sequence-specific DNA-binding proteins
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%
Overview: Every step of transcription can be regulated
NTPs
KB
R+P
RPc
initial
binding
Kf
Abortive
Initiation
RPo
Elongating
Complex
“isomerization”
Negative control: repressors prevent RNAP binding
R
-35
-10
Positive control: activators facilitate RNAP binding-favorable protein-protein contacts
Favorable
contact
A
*
RNAP holo
-35
-10
Construction of an effective activation system
Activating transcription initiation at KB
(initial binding) step
Activators ( e.g. CAP); facilitate RNAP binding with favorable proteinprotein contact
Favorable
contact
A
*
RNAP holo
-35
∆ G = RT lnKD
if * nets 1.4 kcal/mol, KB goes up 10-fold
-10
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
a-CTD
3. Isolate activator-non-responsive
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?
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
EMBO J (1990) 9:973-979
Slide 42.
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 1 round of transcription (~10-20 molecules)
Full dissociation: 6 min to find site again
MBOC: 509-27
Slide 43
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
R P
O
lacZ
lacY
Permease
Science 2008 322: 442-446
lacA
Repressor
(imports inducer)
Permease-YFP
Combinatorial control of gene expression
AND Logic;
e.g. arabinose operon
AND NOT Logic,
e.g. lac operon
AND NOT logic is used to regulate how E. coli responds to lactose
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)
Additional slides
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
A. N. Kapanidis et al., Science 314, 1144 -1147 (2006)
Initial transcription involves DNA scrunching
Open complex
Abortive initiation complex
Higher E* in Abortive initiation complex than open
complex results from DNA scrunching
Initial transcription involves DNA scrunching
Open complex
Abortive initiation complex
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
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 weak operators significantly enhance represssion
EMBO J (1990) 9:973-979
Coherent feed-forward loop allows timing of responses
Example: response to sugars
Transient input
CAP-cAMP
MalT activator
Sustained input