Single-molecule detection of DNA transcription and replication

Transcription initiation by RNA polymerase

Topology of promoter unwinding Lk = Tw + Wr = const promoter RNAP  Tw = -1  Wr = +1

Observation of promoter unwinding by bacterial RNA polymerase Positively supercoiled DNANegatively supercoiled DNA Promoter unwinds DNA extension increases Promoter unwinds DNA extension decreases

Calibration of DNA supercoiling In linear regime (II)  l = 56 nm/turn “plectoneme”

Direct observation of promoter unwinding: consensus lac promoter  l obs,-  l obs,+

Positively supercoiled DNA containing three lac(cons) promoters in tandem  three bubbles  0  1  2  3

More Control Experiments 2. No promoter unwinding is observed in the absence of the initiation factor  ; 3. No unwinding is observed at temperatures below 23 C; 4. Unwinding is abolished by prior addition of heparin (binds free RNAP); 1. No unwinding is observed with a DNA template having no promoter;

Analysis of transition amplitudes (  l obs-,  l obs+ ) Why is the transition amplitude greater for positively supercoiled DNA ??  l obs,- = 50 nm  l obs,+ = 80 nm

 l u = 65 nm  unwinding = 13 bp;  = 15 nm  bend = 110 o …what if RNAP bends the promoter DNA? A bend will always lead to a decrease  in DNA extension  l obs,- +  l obs,+ 2  l u =  =  l obs,- -  l obs,+ 2  l obs : observed signal  l u : signal to due unwinding  : signal due to bending

“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 Lifetime T unwound = 1/k r is concentration-independent T wait T unwound Waiting time T wait = 1/k f depends linearly on inverse concentration (TAU plot)

What does concentration-dependence tell us? PROMOTER RNAP PROMOTER RNAP K B = 100 nM -1 K f = 0.3 s -1 RNAP K r = s -1

T unwound T wait 23°C 25°C 28°C 34°C 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 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 T unwound lifetime, s density of supercoiling,  T wait Torque Increases ( I ) Torque is constant ( II ) 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  A critical torque must be reached for supercoils to form. Torque begins to saturate as supercoils form (  denat ~5 pN nm) Constant extension (zero) Force varies with  Supercoils form early Torque increases with supercoiling Torque saturates when DNA denatures (  denat ~ -0.06,  denat ~8 pN nm) Extended Single molecule “In vivo”: circular plasmid

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  M ppGpp.

2 mM initiating nucleotides stabilizes open promoter (lacCONS) cgtataatgtgtggAAtt no NTP ATP UTP CTP GTP

2 mM initiating nucleotide stabilizes open promoter (rrnB P1) ctataatgcgccaccActg

Observation of promoter clearance: rationale positively supercoiled template real time DNA extension +NTPs

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)

Rates are (essentially) independent of force Wang et al., Nature (1998)

High Stall forces are observed Wang et al., Nature (1998)

RNA Polymerase tracks the DNA axis Harada et al., Nature (2001)

DNA Polymerases Processivity low in the absence of “processivity factors”  need a different scheme Maier et al., PNAS (2000) 97:

DNAp converts ssDNA to (stiffer) dsDNA Maier et al., PNAS (2000) 97:

DNA replication rate is force-dependent Maier et al., PNAS (2000) 97:

Force-dependence results (con’t) Maier et al., PNAS (2000) 97:

Observation of T7 DNAp exonuclease activity Wuite et al., Nature (2000) 404:

Acknowledgements Rutgers Univ. A. Revyakin R.H. Ebright Research on transcription initiation funded by the Cold Spring Harbor Fellows program