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Single-molecule detection of DNA transcription and replication. Transcription initiation by RNA polymerase. D Wr = +1. promoter. RNAP. Topology of promoter unwinding. Lk = Tw + Wr = const. D Tw = -1. Observation of promoter unwinding by bacterial RNA polymerase. Negatively supercoiled DNA.

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topology of promoter unwinding

DWr = +1

promoter

RNAP

Topology of promoter unwinding

Lk = Tw + Wr = const

DTw = -1

observation of promoter unwinding by bacterial rna polymerase
Observation of promoter unwinding by bacterial RNA polymerase

Negatively supercoiled DNA

Positively supercoiled DNA

Promoter unwinds

Promoter unwinds

DNA extension decreases

DNA extension increases

calibration of dna supercoiling
Calibration of DNA supercoiling

In linear regime (II)

dl = 56 nm/turn

“plectoneme”

positively supercoiled dna containing three lac cons promoters in tandem three bubbles

 0

 1

 2

 3

Positively supercoiled DNA containing three lac(cons) promoters in tandem  three bubbles
slide8

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 d l obs d l obs
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

Dlobs,-+ Dlobs,+

Dlu =

2

Dlobs,-- Dlobs,+

e =

2

…what if RNAP bends the promoter DNA?

A bend will always lead to a decreasee 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

waiting times lifetimes obey single exponential statistics
“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
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
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

slide14

Twait

Tunwound

Twait

Tunwound

Twait

Tunwound

Twait

Tunwound

Temperature-dependence in agreement

with bulk results

23°C

25°C

28°C

34°C

supercoiling dependence of promoter unwinding
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

slide17

100

Twait

80

60

lifetime, s

Torque

Increases

(I)

Torque

is constant

(II)

40

20

Tunwound

0

0.5

1

1.5

2

2.5

density of supercoiling, %

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.

does torque saturate in vivo
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)

Does torque saturate in vivo?

Extended Single molecule

“In vivo”: circular plasmid

effect of inhibitor nucleotide ppgpp on lifetime of open promoter complex
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.

2 mm initiating nucleotides stabilizes open promoter laccons

-10

+1

cgtataatgtgtggAAtt

2 mM initiating nucleotides stabilizes open promoter (lacCONS)

no NTP

ATP

UTP

CTP

GTP

slide22

DNA extension

real time

+NTPs

Observation of promoter clearance: rationale

positively supercoiled template

ot measurements of elongation rate
OT measurements of elongation rate

Wang et al., Nature (1998) 282 902-907

rates are essentially independent of force
Rates are (essentially) independent of force

Wang et al., Nature (1998) 282 902-907

high stall forces are observed
High Stall forces are observed

Wang et al., Nature (1998) 282 902-907

rna polymerase tracks the dna axis
RNA Polymerase tracks the DNA axis

Harada et al., Nature (2001) 409 113-115

dna polymerases
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
DNAp converts ssDNA to (stiffer) dsDNA

Maier et al., PNAS (2000) 97: 12002-12007

dna replication rate is force dependent
DNA replication rate is force-dependent

Maier et al., PNAS (2000) 97: 12002-12007

force dependence results con t
Force-dependence results (con’t)

Maier et al., PNAS (2000) 97: 12002-12007

observation of t7 dnap exonuclease activity
Observation of T7 DNAp exonuclease activity

Wuite et al., Nature (2000) 404: 103-106

acknowledgements
Acknowledgements

Rutgers Univ.

A. Revyakin

R.H. Ebright

Research on transcription initiation funded by the

Cold Spring Harbor Fellows program

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