DNA-dependent RNA synthesis or transcription is a complex process that occurs in a series of stages including, transcription initiation, elongation, and termination. During transcription initiation the RNA polymerase locates and binds to a promoter and initiates RNA synthesis at a specific site on the promoter. Transcription initiation is therefore the first important step at which gene expression is regulated. In contrast to bacterial and eucaryotic RNA polymerases that require several accessory proteins to catalyze specific initiation, the bacteriophage RNA polymerases such as T7, T3, and SP6 have the remarkable ability of catalyzing specific initiation without any accessory proteins. These single-subunit RNA polymerases therefore provide an ideal system to investigate the structural, thermodynamic, and kinetic basis for the specificity of transcription initiation. The long term goal of this research proposal is to understand the mechanism of transcription initiation catalyzed by T7 RNA polymerase and to determine how the polymerase modulates its transcriptional efficiency at various T7 promoters. To understand the mechanism of transcription initiation, we propose to delineate kinetic pathway of RNA synthesis. The kinetic studies will include identifying various steps during initiation and measuring their intrinsic rate constants and equilibrium constants. This will be accomplished by studying protein-DNA interactions using quantitative equilibrium binding experiments, and by measuring the single- turnover kinetics of RNA synthesis using stopped-flow and rapid quench- flow kinetic approaches. These studies will be carried out with both strong and weak T7 promoters to understand the mechanistic basis for the modulation of the transcriptional efficiency of T7 RNA polymerase. Transcription by T7 RNA polymerase is also negatively regulated by the T7 lysozyme protein. It has been shown that T7 lysozyme forms a specific complex with T7 RNA polymerase which results in the inhibition of transcription. We will investigate the inhibition mechanism by measuring the kinetic constants of each step during initiation and elongation in the presence of T7 lysozyme. Both strong and weak promoters will be studied to elucidate the differential effects of lysozyme on these promoters.
Showing the most recent 10 out of 36 publications
