Promoter unwinding and promoter escape by RNA polymerase...
Transcript of Promoter unwinding and promoter escape by RNA polymerase...
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Promoter unwinding and promoter escapeby RNA polymerase:
analysis by single-molecule DNA nanomanipulation
Andrei Revyakinand Richard H. EbrightHoward Hughes Medical Institute, Rutgers University
Terence StrickInstitut Jacques Monod
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• primary regulated step in gene expression
backgroundtranscription
• synthesis of an RNA copy of genetic information in DNA
• first step in gene expression
• target of ansamycin-class antibacterial agents (e.g., rifampicin)
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• transcription terminationRNA polymerase dissociates from DNA and releases the RNA molecule
backgroundtranscription
• transcription initiationRNA polymerase binds to DNA and begins synthesis of an RNA molecule
• transcription elongationRNA polymerase translocates along DNA and extends the RNA molecule
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promoter unwinding promoter escape
backgroundtranscription initiation
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experimental approach
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experimental approachexperimental setup (see Strick et al., 1996)
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experimental approachexperimental setup (see Strick et al., 1996)
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experimental approachcalibration curve
DN
Aex
tens
ion,
μm
Positive supercoilingNegative supercoiling
Magnet turns, n
60 nm60 nm
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experimental approachmonitoring DNA unwinding by monitoring bead movement
Lk = Tw + WrLk = Tw + Wr = constant
paramagnetic bead
glass surface
unwinding of DNA double helixby n turns
formation of n positive supercoils
change in position of bead by by n60 nm
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experimental approachmonitoring DNA unwinding by monitoring bead movement
promoter
positivesupercoiling RNAP n positive
supercoilsadded
Bead goes down ~n60 nm
promoternegativesupercoiling
n negativesupercoilsremoved
Bead goes up ~n60 nm
RNAP
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promoter unwinding
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promoter unwinding promoter escape
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promoter unwindingdetection
posi
tivel
y su
perc
oile
d D
NA
nega
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d D
NA
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• No unwinding is observed in the absence of σ.
• No unwinding is observed at low temperatures.
• No unwinding is observed in the absence of RNAP.
• Unwinding is prevented by prior addition of heparin.
• Unwinding is not affected by subsequent addition of heparin.
• The number of unwinding events observed equals the number of promoters. (One unwinding event is observed on a DNA template having one promoter. Two unwinding events are observed on a DNA template having two promoters. Three unwinding events are observed on a DNA template having three promoters.)
promoter unwindingcontrol experiments
• No unwinding is observed in the absence of a promoter.
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DN
Aex
tens
ion
(μm
)
number of intermediates: no detectable intermediates
unwinding: 1.3 turn = 14 bpcompaction: 15 nm
stability of unwound complexk-2: 0.025 s-1
rate of formation of unwound complexKB: 1 x 107 M-1
k2: 1 s-1
R+P ← RPc ← RPo→ →KB k2
k-2
.
.states
• number of states number of intermediates
amplitude
• amplitude of change in DNA extension extent of unwinding and compaction
Twait
• time interval between events (Twait) rate of formation of unwound complex
Tunwound
• lifetime of unwound complex (Tunwound) stability of unwound complex
promoter unwindingobservables, results
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promoter unwinding: effects of supercoiling
Twait
degree of supercoiling, σ
time,
s
Positive supercoiling decreases the rate offormation of the unwoundcomplex.
promoter unwinding: effects of supercoilingrate of formation of the unwound complex (Twait)
consensus promoter
positivelysupercoiled DNA
+5 turns
+7 turns
+9 turns
Twait
Twait
Twait
DN
A e
xten
sion
, μm
DN
A e
xten
sion
, μm
DN
A e
xten
sion
, μm
variable-torqueregime
constant-torqueregime
Effect is due to torque.
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promoter unwinding: effects of supercoilingstability of the unwound complex (Tunwound)
Positive supercoiling decreases the stabilityof the unwound complex.
degree of supercoiling, σ
aver
age
lifet
ime,
s
Tunwound
consensus promoter
positivelysupercoiled DNA
+5 turns
+7 turns
+9 turns
Tunwound
DN
A e
xten
sion
, μm
DN
A e
xten
sion
, μm
DN
A e
xten
sion
, μm
Tunwound
Tunwound
variable-torqueregime
constant-torqueregime
Effect is due to torque.
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negatively supercoiled DNA
irreversible unwinding
DN
A e
xten
sion
, μm Tunwound
positively supercoiled DNA
reversible unwinding
DN
A e
xten
sion
, μm
Tunwound
negatively supercoiled DNA
reversible unwinding
DN
A e
xten
sion
, μm
Tunwound
positively supercoiled DNA
no unwinding
DN
A e
xten
sion
, μm
promoter unwinding: effects of promoter sequence
CTTGACACTTTATGCTTCGGCTCGTATAATGTGTGGA
consensusCTTGTCAGGCCGGAATAACTCCCTATAATGCGCCACCA
rrnB P1
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promoter unwinding: effects of ppGpp
Tunwound, s
inte
grat
ed p
roba
bilit
y
ppGpp absentTunwound = 29 s
consensus
positivelysupercoiled DNA
100 μM ppGpp
ppGpp presentTunwound = 9 s
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promoter unwinding: effects of initiating nucleotideIn
tegr
ated
pro
babi
lity consensus
positivelysupercoiled DNA
2 mM ATP
Tunwound, s
ATP presentTunwound = 400 s
ATP absentTunwound = 40 s
cgtataatgtgtggAatt
+1-10
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promoter unwindingsummary
• We have developed a DNA-nanomanipulation assay for promoter unwinding
• We have applied the assay to assess effects of supercoiling, sequence,effectors, and nucleotides
• The assay permits determination of the number of unwinding intermediates, extent of unwinding, extent of compaction, and kinetics (KB, k2, k-2)
• Supercoiling influences promoter unwinding through mechanical effects (torque), not through structural effects (position or number of supercoil plectonemes)
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promoter escape
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rationaledetection of promoter escape
unwinding event 3
unwinding event 2
unwinding event 1
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detection of promoter escapedata
Time, s
DN
A e
xten
sion
, μm
unwindingevent 1
(RNAP1)
unwindingevent 2
(RNAP1 + RNAP2)
unwinding event 1
unwinding event 2
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kclearR+P ← RPc ← RPo ← RDe
→ →KB k2
k-2
kclear→
Time, s
DN
A e
xten
sion
, μm
unwinding event 1
unwinding event 2
states..
number of unwinding intermediatesin unwinding event 2
• number of statesin unwinding event 2
amplitude
extent of unwinding and compactionin elongation complex
• amplitude of change in DNA extensionin unwinding event 2
Twait-2
Tclear + Twait• time interval between unwinding event 1 and unwinding event 2 (Twait-2)
detection of promoter escapeobservables, results
unwindingevent 1
(RNAP1)
unwindingevent 2
(RNAP1 + RNAP2)
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detection of promoter escapesummary
• We have developed a DNA-nanomanipulation assay for promoter escape
• The assay permits determination of the number of unwinding intermediates in promoter clearance, extent of unwinding in elongation, extent of compaction in elongation, and kinetics (kclear)
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objectives
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• development of DNA-nanomanipulation assay for elongation
objectives
• systematic analysis of effects of activators
applications
• systematic analysis of effects of small-molecule inhibitors
• systematic analysis of ATP-dependent, TFIIH-dependent promoter melting and promoter clearance by eukaryotic RNA polymerase II
• optimization of DNA-nanomanipulation assay for promoter escape
methods development
• development of DNA-nanomanipulation assay for termination
• improvement of temporal resolution
• integration of DNA-nanomanipulation and single-molecule-fluorescence assays
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objectivesDNA-nanomanipulation and single-molecule fluorescence
• simultaneous monitoring of DNA unwinding and RNAP binding(RNAP* imaging)
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• simultaneous monitoring of DNA unwinding and RNAP binding(RNAP* imaging)
objectivesDNA-nanomanipulation and single-molecule fluorescence
• simultaneous monitoring of DNA unwinding and NTP binding(RNAP*-NTP* FRET)
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• simultaneous monitoring of DNA unwinding and RNAP binding(RNAP* imaging)
objectivesDNA-nanomanipulation and single-molecule fluorescence
• simultaneous monitoring of DNA unwinding and NTP binding(RNAP*-NTP* FRET)
• simultaneous monitoring of DNA unwinding and RNAP position(RNAP*-DNA* FRET)
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• simultaneous monitoring of DNA unwinding and RNAP binding(RNAP* imaging)
objectivesDNA-nanomanipulation and single-molecule fluorescence
• simultaneous monitoring of DNA unwinding and NTP binding(RNAP*-NTP* FRET)
• simultaneous monitoring of DNA unwinding and RNAP position(RNAP*-DNA* FRET)
• simultaneous monitoring of DNA unwinding and RNAP conformation(RNAP*/* FRET)
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nega
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d D
NA
posi
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perc
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d D
NA
Promoter unwinding and promoter escapeby RNA polymerase:
analysis by single-molecule DNA nanomanipulation
Andrei Revyakin and Richard H. EbrightHoward Hughes Medical Institute, Rutgers University
Terence StrickInstitut Jacques Monod
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torqueconstantwith +σ
supercoilingdoes not affect
unwinding
torqueconstantwith -σ
supercoilingdoes not affect
unwinding
torqueincreaseswith +σ
supercoilingaffects
unwinding
torqueincreases
with -σ
supercoilingaffects
unwinding
calibration curve: DNA extension versus number of turns
Positive supercoilingNegative supercoiling
DN
Aex
tens
ion,
μm
Magnet turns, n