The objective of this project is to understand control of the elongation phase of RNA synthesis by RNA polymerase, a critical facet of transcription regulation. We will utilize a specific antiterminator protein, the Q protein of bacteriophage lambda, which becomes a subunit of RNA polymerase at a genome-specific site and thereby allows expression of downstream genes by preventing transcription termination. This protein provides a highly defined and accessible system with which to obtain mechanistic insights into universal enzymatic processes that control transcription elongation;for example, the TAT protein of the HIV virus is a regulator of transcription elongation that acts in many ways like lambda Q protein and other bacterial antiterminators. A detailed understanding of these mechanisms will allow approaches to therapies that depend upon the design of specific molecular agents. We will learn the nature of a structural modification, named a barrier that lambda Q protein induces in RNA polymerase in order to make it insensitive to terminators. We will study the role of the transcription elongation factor NusA in constructing the barrier, through biochemical and genetic analysis. We will use mutational analysis to discover the sites and pathways of modification of RNA polymerase by lambda Q protein, in particular to understand how Q regulates transcription pausing, a universal functional behavior of RNA polymerase. This work will complement our continuing efforts to understand the mechanism of termination itself. We also will study the mechanism of action of the protein Mfd, which mediates the process of transcription-coupled DNA repair, and acts to dissociate stalled elongation complexes as it recruits DNA repair proteins. Understanding how the energy of ATP is used by Mfd will illuminate the mechanism of termination and the energetic barriers involved in termination.
Using bacterial and bacteriophage model systems, this project contributes to understanding basic mechanisms of genetic regulation, an undertaking essential to discovering the molecular basis of disease. It also directly investigates regulatory pathways related to those essential to pathogenesis by HIV and infection by toxic bacteria.
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