Singlemolecule detection of DNA transcription and replication

1
Single-molecule detection ofDNA transcription
and replication
2
Transcription initiation by RNA polymerase
3
Topology of promoter unwinding
Lk Tw Wr const
DTw -1
4
Observation of promoter unwinding by bacterial
RNA polymerase
Positively supercoiled DNA
Negatively supercoiled DNA
Promoter unwinds
Promoter unwinds
DNA extension decreases
DNA extension increases
5
Calibration of DNA supercoiling
In linear regime (II) dl 56 nm/turn
plectoneme
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Direct observation of promoter unwinding
consensus lac promoter
Dlobs,-
Dlobs,
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Positively supercoiled DNA containing three
lac(cons) promoters in tandem ? three bubbles
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More Control Experiments
1. No unwinding is observed with a DNA
template having no promoter
2. No promoter unwinding is observed in the
absence of the initiation factor s
3. No unwinding is observed at temperatures
below 23 C
4. Unwinding is abolished by prior addition of
heparin (binds free RNAP)
9
Analysis of transition amplitudes (Dlobs- ,
Dlobs)
Dlobs,- 50 nm
Dlobs, 80 nm
Why is the transition amplitude greater for
positively supercoiled DNA ??
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what if RNAP bends the promoter DNA?
A bend will always lead to a decrease e in DNA
extension
Dlobs observed signal Dlu signal to due
unwinding e signal due to bending
Dlu 65 nm ? unwinding 13 bp e 15 nm ? bend
110o
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Waiting times lifetimes obey
single-exponential statistics
Time-intervals between formation of open complex
Lifetime of open complex
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Concentration-dependence of rate of formation
and dissociation of open promoter complex
Twait
Tunwound

• Lifetime Tunwound 1/kr is concentration-independ
  ent

• Waiting time Twait 1/kf depends linearly on
  inverse concentration (TAU plot)

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What does concentration-dependence tell us?
RNAP
PROMOTER
KB 100 nM-1
RNAP
PROMOTER
Kf 0.3 s-1
RNAP


Kr 0.025 s-1
RNAP
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Temperature-dependence in agreement with bulk
results
23C
25C
28C
34C
15
Effects of promoter sequenceunwinding at the
rrnB P1 promoter
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Supercoiling-dependence of promoter unwinding
lac(cons)
rrnB P1
Positive supercoiling slows down formation of
o.c. and destabilizes o.c.
Equilibrium shifts 15-fold for an increase in
supercoiling density of 0.007
Negative supercoiling stabilizes o.c.
A supercoiling-dependent regime is followed by a
supercoiling-independent regime
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Formation of open-promoter complex is highly
sensitive to DNA torque
Torque increases by about 0.2 pN nm/turn for
data in regime (I) and saturates at about 5 pN nm.
18
Does torque saturate in vivo?
Extended Single molecule
In vivo circular plasmid

• Constant force
• Extension varies with s
• A critical torque must be reached for supercoils
  to form.
• Torque begins to saturate as supercoils form
  (Gdenat5 pN nm)

• Constant extension (zero)
• Force varies with s
• Supercoils form early
• Torque increases with supercoiling
• Torque saturates when DNA denatures
• (sdenat -0.06, Gdenat8 pN nm)

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Effect of inhibitor nucleotide ppGppon lifetime
of open promoter complex
A 3-fold destabilization (from 30s to 10s) of
open-promoter lifetime is observed at both
promoters upon addition of 100 mM ppGpp.
20
2 mM initiating nucleotides stabilizes open
promoter (lacCONS)
no NTP
ATP
UTP
CTP
GTP
21
2 mM initiating nucleotide stabilizes open
promoter (rrnB P1)
22
Observation of promoter clearance rationale
positively supercoiled template
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Transcription observed with all 4 nucleotides
(I) control experiment (sc lac promoter)
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Transcription observed with all 4 nucleotides (II)
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OT measurements of elongation rate
Wang et al., Nature (1998) 282 902-907
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Rates are (essentially) independent of force
Wang et al., Nature (1998) 282 902-907
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High Stall forces are observed
Wang et al., Nature (1998) 282 902-907
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RNA Polymerase tracks the DNA axis
Harada et al., Nature (2001) 409 113-115
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DNA Polymerases
Processivity low in the absence of processivity
factors ? need a different scheme
Maier et al., PNAS (2000) 97 12002-12007
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DNAp converts ssDNA to (stiffer) dsDNA
Maier et al., PNAS (2000) 97 12002-12007
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DNA replication rate is force-dependent
Maier et al., PNAS (2000) 97 12002-12007
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Force-dependence results (cont)
Maier et al., PNAS (2000) 97 12002-12007
33
Observation of T7 DNAp exonuclease activity
Wuite et al., Nature (2000) 404 103-106
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Acknowledgements
Rutgers Univ. A. Revyakin R.H. Ebright
Research on transcription initiation funded by
the Cold Spring Harbor Fellows program