Mechano-Chemical Kinetics and Mechanism of Transcription

Cornell University, Ithaca NY

Investigators

Abstract

Transcription is the first step in gene expression and a major point of gene regulation. It is carried out by RNA polymerase (RNAP) which functions as a highly processive molecular motor that moves along a DNA template transcribing genetic information from DNA to messenger RNA. The mechano-chemical mechanism by which RNAP carries out transcription remains one of the most fundamental and intriguing problems in biology. We will carry out a comprehensive set of single molecule experiments to probe the chemical and mechanical behaviors of E. coli RNAP. Single molecule mechanical studies offer the promise of probing the kinetics of transcription by tracking motions of individual molecules. This eliminates the necessity of synchronization of a population of molecules, and makes it possible to differentiate transcription pausing vs. active elongation. Furthermore, translocations of RNAP can be probed directly by the application of an external load that facilitates or hinders mechanical movements. These experiments, in conjunction with theoretical modeling, will help to differentiate between competing possible mechanisms of transcription: power-stroke vs. thermal ratchet. Although the project focuses on E. coli RNAP, the general methodology and approach outlined are readily extendable to many other DNA-based molecular motors. This combination of biochemical techniques, single molecule measurements, and theoretical modeling should prove to be a powerful cross-disciplinary approach for tackling important problems in biology and will provide cross disciplinary training to undergraduate and graduate students. The results of the project will be well disseminated through publications, presentations, establishment of a transcription modeling website, and broader education to the community via a local science museum.