Protein/Protein Interactions in Prokaryotic Gene Control

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DESCRIPTION (provided by applicant): Transcription in bacteria depends on a multi-subunit RNA polymerase (RNAP) that is conserved from bacteria to man. The study of prokaryotic systems has defined the basic transcription cycle, providing a mechanistic framework for the study of transcription in all organisms. Because RNAP is the primary target of gene regulation, an in depth understanding of its structure and function is essential for elucidating the variety of regulatory mechanisms that control gene expression. The principles that emerge from investigations of the transcription apparatus and its regulation in bacterial systems continue to drive both the development of new strategies to control microbial pathogens and the study of the many transcription-based processes that underlie human development and disease processes. For transcription to initiate in bacteria, the catalytically proficient core enzyme must combine with ? factor to form the holoenzyme. In addition to the roles of ? factors in transcription initiation, which have been intensively investigated over many years, accumulating evidence indicates that ? factors can participate in events downstream of initiation and, moreover, that regulators can target the RNAP holoenzyme during elongation. This has been established for the Q antiterminator protein of bacteriophage ?, which engages the RNAP holoenzyme at a ?-dependent early elongation pause. The first aim of the proposed research is to investigate Q's interactions with the RNAP holoenzyme during early elongation to understand the nature of the specialized pathway by which Q gains access to and alters the behavior of the transcription complex. The second aim of the proposed research is to investigate a ?/core interaction that influences the functional properties of RNAP in a stage-specific manner, modulating both promoter escape and early elongation pausing. The third aim of the proposed research is to investigate an unexpected role for a ? factor as a transcription antiterminator and to investigate how the presence of ? in mature elongation complexes might be regulated. Together, the experiments proposed in Aims 2 and 3 will lead to a deeper understanding of the dynamic roles played by ? factors throughout the transcription cycle.