Force and Velocity Measured for Single Molecules of RNA Polymerase

1. Force and Velocity Measured for Single Molecules of RNA Polymerase Michelle D. Wang, Mark J. Schnitzer, Hong Yin, Robert Landick, Jeff Gelles, Steven M. Block M. D. Wang and S. M. Block, Department of Molecular Biology and Princeton Materials Institute, Princeton University, Princeton, NJ 08544, USA. M. J. Schnitzer, Departments of Physics and Molecular Biology, Princeton University, Princeton, NJ 08544, USA. H. Yin and J. Gelles, Department of Biochemistry, Brandeis University, Waltham, MA 02254, USA. R. Landick, Department of Bacteriology, University of Wisconsin, Madison, WI 53706, USA. Science 30 October 1998, Vol. 2, pp. 902-907.

2. Key Points & Facts The relationship between applied force F and steady-state velocity V is a fundamental characteristic of the enzyme mechanism itself. • F-V relationships have been determined for three biological motors: • ensembles of myosin in contracting muscles, • single molecules of kinesin moving along microtubules, • and the rotary engine that spins bacterial flagella. Force and Velocity Measuremed for Single Molecules of RNA Polymerase

3. The Actual Setup Force and Velocity Measuremed for Single Molecules of RNA Polymerase

4. Alternative Setups Force and Velocity Measuremed for Single Molecules of RNA Polymerase

5. Force Affects Translocation Force and Velocity Measuremed for Single Molecules of RNA Polymerase

6. 2-5 nm/s Default strain needed Elasticity of DNA causes ~50 nm jump Trap stiffness proportional to force Open- and Closed-Loop Trapping Modes Force and Velocity Measuremed for Single Molecules of RNA Polymerase

7. Transcription Stall Forces • Transcription stalled at trap stifnesses of 0.25 and 0.29 pN/nm • This corresponds to a force of 30 to 35 pN • Previously reported 14 pN (1995, ref. 2) • ~20 % of beads were not stopped • Irreversible stalls caused by prolonged exposure to laser light • Stalls did not occur in a homogeneous fasion • => Stall force might be a function of nucleotide sequence • New setup (not even optimized) improves the following: • Photodamage is minimized with the feedback loop • Higher peak powers can be achieved (stronger traps) • Dynamic response of the system improved • Force can be recorded in ms • RNAP can be stopped within seconds (5-40 fold faster) Force and Velocity Measuremed for Single Molecules of RNA Polymerase

8. Transcription Stall Force In the presence of saturating NTPs and 1 µM PPi, the stall force was 25 pN. Raising the pyrophosphate (PPi)concentration to 1 mM slowed the mean elongation rate at low force by 2.3-fold and yielded a stall force of 23 pN, which is not significantly different. This change reduces the estimated free energy for the RNAP condensation reaction by mass action and the fraction of free energy converted into mechanical work near stall is estimated at 44% for 1 mM PPi(and18% at 1 µM PPi) whichresembles kinesin that spends roughly half its available free energy as mechanical work near stall. Force and Velocity Measuremed for Single Molecules of RNA Polymerase

9. Force and Velocity Measurements 1 bp = 0.338 nm Low-load => no change High-load => Stall! ”Once trap properties are calibrated and adjustments are made for series compliance, it is possible to convert measurements of bead displacement and trap stiffness directly into records of time-varying force and RNAP position along the template, and thereby into RNA transcript length” Force and Velocity Measuremed for Single Molecules of RNA Polymerase

10. Characteristic Load Distance (5-10 bps) Force-Velocity Relationships for RNAP v, a dimensionless velocity (normalized to the unloaded speed V0)and f, a dimensionless force (normalized to the force at halfmaximal velocity F1/2), before averaging. Force and Velocity Measuremed for Single Molecules of RNA Polymerase

11. Comparisons with Theory • Stalling is an elongation-incompetent state • RNAP slides backwards (5-10 bps) • Maintains register between DNA and RNA • Resumes transcription after reduction of load • RNAP moves bidirectionaly through a distance corresponding to 5-10 bps • Similar for these models is: • Reaction schemes are tightly coupled • One condensation reaction per bp • Involve large-scale movement of the RNAP associated with stalling • Large drop in velocity upon stall is incompatible with single bp load-stepping • The rate limiting transition is not load-dependent over most of the force range • The F-V curves are convex Force and Velocity Measuremed for Single Molecules of RNA Polymerase

12. Force and Velocity Measuremed for Single Molecules of RNA Polymerase

13. Force and Velocity Measuremed for Single Molecules of RNA Polymerase