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

Friday the 14th of November 2008Force and Velocity Measuremed for Single Molecules of RNA Polymerase2 Key Points & Facts F-V relationships have been determined for three biological motors: 1.ensembles of myosin in contracting muscles, 2.single molecules of kinesin moving along microtubules, 3.and the rotary engine that spins bacterial flagella. The relationship between applied force F and steady-state velocity V is a fundamental characteristic of the enzyme mechanism itself.

Friday the 14th of November 2008Force and Velocity Measuremed for Single Molecules of RNA Polymerase3 The Actual Setup

Friday the 14th of November 2008Force and Velocity Measuremed for Single Molecules of RNA Polymerase4 Alternative Setups

Friday the 14th of November 2008Force and Velocity Measuremed for Single Molecules of RNA Polymerase5 Force Affects Translocation

Friday the 14th of November 2008Force and Velocity Measuremed for Single Molecules of RNA Polymerase6 Open- and Closed-Loop Trapping Modes 2-5 nm/s Default strain needed Trap stiffness proportional to force Elasticity of DNA causes ~50 nm jump

Friday the 14th of November 2008Force and Velocity Measuremed for Single Molecules of RNA Polymerase7 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) Transcription Stall Forces

Friday the 14th of November 2008Force and Velocity Measuremed for Single Molecules of RNA Polymerase8 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 (and 18% at 1 µM PPi) which resembles kinesin that spends roughly half its available free energy as mechanical work near stall.

Friday the 14th of November 2008Force and Velocity Measuremed for Single Molecules of RNA Polymerase9 Force and Velocity Measurements ”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” 1 bp = nm Low-load => no changeHigh-load => Stall!

Friday the 14th of November 2008Force and Velocity Measuremed for Single Molecules of RNA Polymerase10 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. Characteristic Load Distance (5-10 bps)

Friday the 14th of November 2008Force and Velocity Measuremed for Single Molecules of RNA Polymerase11 Comparisons with Theory 1. Stalling is an elongation-incompetent state RNAP slides backwards (5-10 bps) Maintains register between DNA and RNA Resumes transcription after reduction of load 2.RNAP moves bidirectionaly through a distance corresponding to 5-10 bps Similar for these models is: 1.Reaction schemes are tightly coupled One condensation reaction per bp 2.Involve large-scale movement of the RNAP associated with stalling Large drop in velocity upon stall is incompatible with single bp load-stepping 3.The rate limiting transition is not load-dependent over most of the force range The F-V curves are convex

Friday the 14th of November 2008Force and Velocity Measuremed for Single Molecules of RNA Polymerase12

Friday the 14th of November 2008Force and Velocity Measuremed for Single Molecules of RNA Polymerase13