In this fellowship application I propose to use scanning force microscopy (SFM) methods to investigate the mechanical translocation of individual T7 phage RNA polymerase molecules during transcription initiation. Through these studies, I intend to more precisely determine the complex dynamics of transcription as T7 RNA polymerase begins to move along the DNA template and to more fully characterize the relationship between template sequence and initiation efficiency. Specifically I propose: 1. to develop a high resolution SFM-based translocation assay able to directly observe a single nucleotide being added to the nascent RNA chain by T7 RNA polymerase. 2. using this assay, to characterize the nucleotide-by-nucleotide transcription initiation process of a single T7 RNA polymerase molecule.
