Transcript Slide 1

Single-molecule detection of
DNA transcription and replication
Transcription initiation by RNA polymerase
Topology of promoter unwinding
Lk = Tw + Wr = const
DWr = +1
promoter
DTw = -1
RNAP
Observation of promoter unwinding
by bacterial RNA polymerase
Negatively supercoiled DNA
Positively supercoiled DNA
Promoter unwinds
Promoter unwinds
DNA extension increases
DNA extension decreases
Calibration of DNA supercoiling
In linear regime (II)
dl = 56 nm/turn
“plectoneme”
Direct observation of promoter unwinding: consensus lac promoter
Dlobs,Dlobs,+
Positively supercoiled DNA containing three lac(cons)
promoters in tandem  three bubbles
0
1
2
3
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);
Analysis of transition amplitudes (Dlobs- , Dlobs+)
Dlobs,- = 50 nm
Dlobs,+ = 80 nm
Why is the transition amplitude greater for positively supercoiled DNA ??
…what if RNAP bends the promoter DNA?
A bend will always lead to a decrease e in DNA extension
Dlobs : observed signal
Dlobs,-+ Dlobs,+
Dlu Dl
: signal
to due unwinding
u =
e
: signal due 2to bending
e=
Dlobs,-- Dlobs,+
2
Dlu = 65 nm  unwinding = 13 bp; e = 15 nm  bend = 110o
“Waiting” times & lifetimes obey
single-exponential statistics
Time-intervals between formation of
open complex
Lifetime of open complex
Concentration-dependence of
rate of formation and dissociation
of open promoter complex
Twait
Tunwound
• Lifetime Tunwound= 1/kr is concentration-independent
• Waiting time Twait = 1/kf depends linearly on inverse concentration (TAU plot)
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
Twait
23°C
Tunwound
Twait
25°C
Tunwound
28°C
Twait
Tunwound
34°C
Tunwound
Twait
Temperature-dependence in agreement
with bulk results
Effects of promoter sequence:
unwinding at the rrnB P1 promoter
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
Formation of open-promoter complex
is highly sensitive to DNA torque
100
Twait
lifetime, s
80
60
40
Torque
Increases
(I)
Torque
is constant
(II)
20
Tunwound
0
0.5
1
1.5
2
2.5
density of supercoiling, %
Torque increases by about
0.2 pN nm/turn
for data in regime (I) and
saturates at about 5 pN nm.
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
(Gdenat~5 pN nm)
• Constant extension (zero)
• Force varies with s
• Supercoils form early
• Torque increases with
supercoiling
• Torque saturates when
DNA denatures
(sdenat~ -0.06, Gdenat~8 pN nm)
Effect of inhibitor nucleotide ppGpp
on 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.
2 mM initiating nucleotides stabilizes open promoter (lacCONS)
no NTP
ATP
UTP
CTP
GTP
-10
+1
cgtataatgtgtggAAtt
2 mM initiating nucleotide stabilizes open promoter (rrnB P1)
-10
+1
ctataatgcgccaccActg
DNA extension
Observation of promoter clearance: rationale
+NTPs
positively supercoiled template
real time
Transcription observed with all 4 nucleotides
(I) control experiment (+sc lac promoter)
Transcription observed with all 4 nucleotides (II)
OT measurements of elongation rate
Wang et al., Nature (1998) 282 902-907
Rates are (essentially) independent of force
Wang et al., Nature (1998) 282 902-907
High Stall forces are observed
Wang et al., Nature (1998) 282 902-907
RNA Polymerase tracks the DNA axis
Harada et al., Nature (2001) 409 113-115
DNA Polymerases
Processivity low in the absence of “processivity factors”  need a different scheme
Maier et al., PNAS (2000) 97: 12002-12007
DNAp converts ssDNA to (stiffer) dsDNA
Maier et al., PNAS (2000) 97: 12002-12007
DNA replication rate is force-dependent
Maier et al., PNAS (2000) 97: 12002-12007
Force-dependence results (con’t)
Maier et al., PNAS (2000) 97: 12002-12007
Observation of T7 DNAp exonuclease activity
Wuite et al., Nature (2000) 404: 103-106
Acknowledgements
Rutgers Univ.
A. Revyakin
R.H. Ebright
Research on transcription initiation funded by the
Cold Spring Harbor Fellows program