INTER AND INTRAMOLECULAR COMMUNICATIONS IN TRANSCRIPTION

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The major unresolved problem in biology is to understand the mechanisms by which transcription is regulated. Such understanding can be accomplished by establishing structural, thermodynamic and kinetic determinants of regulatory processes. This proposal is focused on structural aspects of regulation of transcription by transcription activators. The model system, which will be used in this work, is E. coli RNA polymerase (RNAP). In our research we will use a combination of fluorescence spectroscopy, protein chemistry and molecular biology, and we will also take advantage of a novel experimental approach developed recently in our laboratory to map protein domains involved in macromolecular interactions. Our long term goals are to understand in structural terms how RNAP, depending on the structure of the promoter and depending on the presence or absence of transcription activators readjusts the set of available interactions to perform its biological function . To achieve our goals we propose: aim #1. Studies on the molecular architecture of activator-RNAP-type I promoter complexes to determine the domains of alpha, sigma and beta subunits involved in protein-protein and protein-DNA interactions, and to determine distances between functional units of the activator-RNAP- promoter complex. aim #2. Studies on the molecular architecture of activator-RNAP-type II promoter complexes to determine differences in overall architecture of complexes with type I and type II promoters, and to formulate a structural model for the activator-RNAP-premoter complex with type I and type II promo rs. aim #3. Studies on the nature of RNAP contact domains to determine their polarity, solvent exposure, flexibility, segmental mobility and communication between them. The results of this study and the novel methodology developed will have general applicability to studies of transcriptional regulation of prokaryotic and eukaryotic systems. Understanding regulation of transcription at the molecular level is of crucial importance for understanding development and the pathogenesis of abnormal development and neoplasia.