Transcription termination and antitermination in E.coli

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DESCRIPTION (provided by applicant) Transcription is key to all the cellular processes and RNA polymerase (RNAP), the enzyme responsible for transcription, is an attractive drug target in different microbial pathogens. At a given time most of the RNAP molecules are engaged in mRNA synthesis and rapid turn over, which involves two crucial steps in transcription, namely elongation and termination. So, ideally drug target should be the DNA-bound RNAP molecules engaged in these two modes, rather than their free form in cytosol. Long term goal of this project is to understand the mechanistic aspects of elongation, termination and as well as antitermination steps of the transcription process, so that a rational drug designing will be possible in future. Major focus will be to elucidate the active site dynamics of RNAP and intricate protein-DNA-RNA interactions during these steps. N-mediated antitermination system from lamdoid phage, which involves E.Coli RNAP, is an ideal system to study the protein-DNA-RNA interactions in the transcription elongation complex and as well as to understand the mechanism of termination/antitermination processes. In vivo studies indicate that shiga-toxin bearing lamdoid phage, H19B, requires a phage factor N and the host factor NusA to modify the elongation complex and achieve antitermination. Therefore, this antitermination complex is much simpler for biochemical and structural studies. Interactions in this modified elongation complex will be characterized by mutagenesis, Fe-BABE cleavage and fluorescence spectroscopy. 3D localization of N-binding surface on RNA polymerase will be obtained from homology modeling based on Taq RNA polymerase and Yeast RNA Pol II crystal structures together with the data obtained from the experiments stated above. In parallel studies, the active-site dynamics of RNAP in response to different DNA sequences, nascent RNA structure and trans factors (like N protein etc.), will be studied experimentally by using, chemical cleavage, foot printing, cross linking, and fluorescence spectroscopy. Computational methods, such as molecular dynamics simulations will also be used to predict the domain movement around the active site, which will be used to design mutations in specific domains and find its interacting partners by suppressor genetics. Understanding of the active site dynamics will lay the foundation for rational drug design in future.