Single molecule analyses of RNA polymerase II elongation

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DESCRIPTION (provided by applicant): The fundamental goal of this project is to establish a method for continuous, real-time analysis of eukaryotic RINA polymerase II transcription through a chromatin template using single-molecule optical trapping techniques. Optical trapping technology permits direct observation of movement by individual RNA polymerase molecules, yielding high-resolution measurement of transcription elongation rates, pausing patterns, and pause durations. This approach allows us to focus on the dynamic interactions that are averaged out during conventional population analyses, providing previously inaccessible information concerning the kinetic aspects of translocation by RNA polymerase and the mechanism by which specific transcription factors regulate this activity. This proposal involves the systematic development of single-molecule techniques specifically tailored for the study of RNA polymerase elongation. Foundational for these analyses is a novel strategy for immobilization of single RNA polymerase molecules without disturbing native elongation activity. Initially, the method will be optimized using an easily manipulable E. coli transcription system, which will serve as a model for establishing single molecule assays of elongation by Drosophila RNA polymerase II. These assays will first be employed to determine the underlying characteristics of RNA polymerase II transcription on naked DNA templates before being exploited for a detailed investigation of the mechanism by which RNA polymerase II navigates through a nucleosomal barrier.