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Single-molecule detection of DNA transcription and replication

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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Single-molecule detection of DNA transcription and replication

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  1. Single-molecule detection ofDNA transcription and replication

  2. Transcription initiation by RNA polymerase

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

  4. Observation of promoter unwinding by bacterial RNA polymerase Negatively supercoiled DNA Positively 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”

  6. Direct observation of promoter unwinding: consensus lac promoter Dlobs,- Dlobs,+

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

  8. 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 ??

  10. 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

  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 Twait Tunwound • Lifetime Tunwound= 1/kr is concentration-independent • Waiting time Twait = 1/kf depends linearly on inverse concentration (TAU plot)

  13. 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

  14. Twait Tunwound Twait Tunwound Twait Tunwound Twait Tunwound Temperature-dependence in agreement with bulk results 23°C 25°C 28°C 34°C

  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. 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.

  18. 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

  19. 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. -10 +1 cgtataatgtgtggAAtt 2 mM initiating nucleotides stabilizes open promoter (lacCONS) no NTP ATP UTP CTP GTP

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

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

  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

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