1. Single-molecule detection of DNA transcription and replication

2. Transcription initiation by RNA polymerase

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

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

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

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

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

8. 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;

9. 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

10.  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

11. “Waiting” times & lifetimes obey single-exponential statistics Time-intervals between formation of open complex Lifetime of open complex

12. 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)

13. What does concentration-dependence tell us? PROMOTER RNAP PROMOTER RNAP K B = 100 nM -1 K f = 0.3 s -1 RNAP K r = 0.025 s -1

14. T unwound T wait 23°C 25°C 28°C 34°C Temperature-dependence in agreement with bulk results

15. Effects of promoter sequence: unwinding at the rrnB P1 promoter

16. 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

17. Formation of open-promoter complex is highly sensitive to DNA torque T unwound lifetime, s density of supercoiling,  00.511.522.5 T wait 20 40 60 80 100 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.

18. 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

19. 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.

20. 2 mM initiating nucleotides stabilizes open promoter (lacCONS) cgtataatgtgtggAAtt +1 -10 no NTP ATP UTP CTP GTP

21. 2 mM initiating nucleotide stabilizes open promoter (rrnB P1) ctataatgcgccaccActg +1 -10

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

23. Transcription observed with all 4 nucleotides (I) control experiment (+sc lac promoter)

24. Transcription observed with all 4 nucleotides (II)

25. OT measurements of elongation rate Wang et al., Nature (1998) 282 902-907

26. Rates are (essentially) independent of force Wang et al., Nature (1998) 282 902-907

27. High Stall forces are observed Wang et al., Nature (1998) 282 902-907

28. RNA Polymerase tracks the DNA axis Harada et al., Nature (2001) 409 113-115

29. DNA Polymerases Processivity low in the absence of “processivity factors”  need a different scheme Maier et al., PNAS (2000) 97: 12002-12007

30. DNAp converts ssDNA to (stiffer) dsDNA Maier et al., PNAS (2000) 97: 12002-12007

31. DNA replication rate is force-dependent Maier et al., PNAS (2000) 97: 12002-12007

32. Force-dependence results (con’t) Maier et al., PNAS (2000) 97: 12002-12007

33. Observation of T7 DNAp exonuclease activity Wuite et al., Nature (2000) 404: 103-106

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