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 ID: 673748
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Slide1
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 6
th
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
factor activity: a structural perspective.
Current Opinion in Micro.
11
:121-127
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:
603-11.Slide2
3.
Studies of Transcription Initiation
Roy S, Lim HM, Liu M,
Adhya
S. (2004) Asynchronous
basepair
openings in transcription initiation: CRP enhances the rate-limiting 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 DNA-scrunching 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:
–DNA Interaction.
Cell
:
147:
1218-1219Slide3
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
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
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
P
RM
promoter. JMB 157: 493-525
d. DNA looping
**
Oehler
, S.,
Eismann
, E.R., Kr
am
er, 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.
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: 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
: 10.1016Slide4
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
: 10.1016Slide5
Important concepts
Cellular RNA polymerases are conserved across all organisms. These important machines not only produce the transcript but also play
regulatory roles.
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.
Slide6
Outline
Introduction to Transcription/RNA polymerase
Bacterial paradigm for transcription
initiation
A. Process of Transcription Initiation
B. Transition to elongation: Abortive Initiation
C. Regulating Transcription
initiationSlide7
The Transcription cycle:
Initiation, Elongation, Termination
Binding: closed complex
Promoter melting: open complex
Initial transcribing complex
Elongation
TerminationSlide8
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)Slide9
Structure of RNAP in t
he
three domains
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
Bacteria
Universally conserved
Archaeal/eukaryotic
Archaea
Eukarya
TranscriptionSlide10
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 eventsSlide11
Transcription initiationSlide12
K
B
K
f
initial
binding
“
isomerization
”
Abortive
Initiation
Elongating
Complex
RP
o
RP
c
R+P
NTPs
Steps in Transcription Initiation
Initiation
transition
Elongation/
terminationSlide13
All cellular RNAPs have initiation subunitsSlide14
The Bacterial paradigm for Initiation
Core RNAP +
sigma
a
2
bb’
s
Holoenzyme
a
2
bb’
s
Initiation factorSlide15
Labmate
Jeff Roberts reported that the new, improved preparation of RNAP (peak 2) had
no activity on
l
DNA
Peak 1 restored activity
Improved purification of RNA polymerase leads to the discovery of
sImproved fractionation
lysate
phosphocellulose column
salt
OD 280
1
2
Activity (*ATP)
CT DNA
Fraction #
SDS gel analysis
Peak 1 Peak 2
'
increases rate of initiation
s
Transcription
DNA
Assay:
incorporation
P
ATP using
l
as templateSlide16
Is the -10 promoter element recognized as Duplex or SS DNA?
-
10 logo
-
35 logo
Helix-turn-helix in Domain 4
Recognizes -35 as duplex DNA
Recognition of the Prokaryotic promoterSlide17
s
is positioned for DNA recognitionSlide18
Transition to elongation:
Abortive initiationSlide19
K
B
K
f
initial
binding
“
isomerization
”
Abortive
Initiation
Elongating
Complex
RP
o
RP
c
R+P
NTPs
Abortive Initiation and Promoter escape
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?
Slide20
Three models for Abortive initiation
#1
Predicts expansion and contraction of RNAP
Predicts expansion and contraction of
DNA
Predicts movement of both the RNAP leading and trailing edge relative to DNA
#2
#3
Science (2006
314:
1139-43; 1144-47;
Slide 38-41Slide21
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
Using single molecule FRET to monitor movement of RNAP and DNA
Conclusion: DNA shortens (scrunching!)Slide22
s
is positioned to block elongating transcripts
In vitro transcription: #1 full-length
s
; #2: truncated
s
: no domain 4 or s3-4 in exit tunnel) Murakami, Darst 2002 Slide23
The Bacterial paradigm
for Regulating
InitiationSlide24
Gene regulation in E. coli: The Broad Perspective
• 3.6
mB
chromosome
4400 genes
7
factors (housekeeping
and alternative
s)
• 300-350 sequence-specific DNA-binding proteins
In E. coli 1 copy/cell ≈ 10
-9
M
If K
D
= 10
-9
M and things are simple:
10 copies/cell 90% occupied
100 copies/cell 99% occupiedSlide25
Overview: Every step of transcription can be regulated
K
B
K
f
initial
binding
“
isomerization
”
Abortive
Initiation
Elongating
Complex
RP
o
RP
c
R+P
NTPs
Negative control: repressors prevent RNAP binding
R
-35
-10
Positive control: activators facilitate RNAP binding-favorable protein-protein contacts
A
-35
-10
RNAP holo
Favorable contact
*Slide26
Construction of an effective activation system
Activating transcription initiation at K
B
(initial binding)
step
∆ G = RT
lnKD if *
nets 1.4 kcal/mol, KB goes up 10-fold
Activators ( e.g. CAP); facilitate RNAP binding with favorable protein-protein contact
A
-35
-10
RNAP holo
Favorable contact
*Slide27
Activating by increasing K
B
is effective only if initial promoter occupancy is low
If favorable contact nets 1.4Kcal/mole (K
B
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
*Slide28
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
3. Isolate activator-non-responsive mutations in
RNAP
-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*
RNAPSlide29
C
onstruction of an effective repression system
-35
-10
Lac operator (
O
1
)
Lac ~ 1980
What is the function of these weak operators?
O
2
1/10 affinity of O
1
O
3
1/300 affinity of O
1
Lac 2000
-35
-10
-90
O
3
O
1
O
2
+400
Oehler, 2000Slide30
OK
O
m
Through DNA looping, Lac repressor binding to a
“
strong
”
operator (O
m
) can be helped by binding to a
“
weak
”
operator (O
A)
O
m
O
a
Better!
M
M
A mutant Lac repressor that cannot form
tetramers is not helped by a weak site
EMBO J (1990) 9:973-979
Slide 42.Slide31
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 1 round of transcription (~10-20 molecules)
Full dissociation: 6 min to find site againSlide32
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
MBOC: 509-27
Slide 43Slide33
Positive feed back loops
Positive feedback loops can generate
bistability
and switch-like responses Slide34
Bistability at the
lac
operon
O
lacZ
lacY
lacA
P
R
Repressor
Permease
(imports inducer)
Permease-YFP
Science 2008
322:
442-446Slide35
Combinatorial control of gene expression
AND NOT Logic, e.g.
lac operon
AND Logic;
e.g. arabinose operonSlide36
AND NOT logic is used to regulate how
E. coli
responds to lactose
Inactive CAP
Active CAP—binds DNA
Regulates >100 genes positively or negatively
cAMP
high glucose
The CAP activator senses nutritional stat
e
O
lacZ
lacY
lacA
P
A
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)
Slide37
Additional slidesSlide38
A. N. Kapanidis et al., Science 314, 1144 -1147 (2006)
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 complexSlide39
Initial transcription involves DNA scrunching
Higher E* in Abortive initiation complex than open complex results from DNA scrunching
Open complex
Abortive initiation complexSlide40
Initial transcription involves DNA scrunching
Open complex
Abortive initiation complexSlide41
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 energy accumulated in the DNA scrunched
“
stressed intermediate could disrupt interactions between RNAP,
and the promoter, thereby driving the transition from initiation to elongationSlide42
The weak operators significantly enhance represssion
EMBO J (1990) 9:973-979Slide43
Coherent feed-forward loop allows timing of responses
Example: response to sugars
Transient input
Sustained input
CAP-cAMP
MalT activator