In vitro transcription is now a routine biochemical technique that allows for the synthesis of RNA molecules from a DNA template. This is done utilizing a variety of bacterial and phage RNA polymerases. Transcriptional pausing is a common biological phenomenon, yet the molecular mechanism of pausing is not yet well understood. Bacterial and phage RNA polymerases have been demonstrated to behave differently when they encounter a protein blockade during transcription. The goal of this project is to characterize the transcription complex as it encounters the protein blockade. Characterization of this complex will enable us to better understand the structural and chemical interactions between the polymerase, the DNA template, and the blockade protein. The protein blockade was engineered using the dCas9 protein from the CRISPR system. By using a nuclease deficient Cas9 protein (dCas9), we can create a protein blockade that will not cleave the template The CRISPR system is an adaptive immune system found in Archaea and many Bacteria. dCas9 protein binds guide RNA that is complementary to a target DNA sequence within the transcription template sequence creating a reversible blockade at specific locations in order to study the RNA polymerase interaction. In vitro conditions for T7 RNA polymerase have been optimized, and initial characterization of the complex has been completed. This work will allow us to better understand the mechanism behind a novel, flexible, and reversible transcriptional control system.
