Structure-based mutational analysis of RNA polymerase

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DESCRIPTION (provided by applicant): Our long-term goal is to understand transcription in molecular detail and to uncover the underlying structural determinants. Transcription, the first step of gene expression and a major regulatory checkpoint, is performed by DNA-dependent RNA polymerases (RNAPs). A crystal structure of Thermus aquaticus (Taq) RNAP core enzyme has recently been determined. The superposition of functional data from E. coli RNAP and structural data from Taq RNAP allowed to put forward hypothetical structure-function models of transcription complex. The models assign specific biochemical functions to several RNAP structural elements. To test predictions of these models, rational mutagenesis of RNAP will be performed. As model systems we will use RNAP from Taq, the only bacterial enzyme for which high-resolution structure is known, and E. co/i RNAP, which is best-studied functionally. Studies of Taq RNAP will make the best use of structural information and are expected to provide new insights in RNAP mechanism and structure. Studies of E. coli enzyme are expected to provide insights in RNAP mechanism and regulation. Genetic systems and biochemical assays to study E. co/i RNAP have been established; there is no genetic system to study Taq RNAP. We developed an E. co/i expression system that allows to overproduce recombinant Taq RNAP with engineered mutations, and we showed that many techniques developed to study E. co/i transcription are applicable to study Taq RNAP. The following are our specific aims. 1) To perform systematic mutagenesis of potentially functionally important structural elements of Tag and E. co/i RNAP Beta and Beta-prime subunits. The following RNAP elements will be targeted initially: the Beta' "rudder", implicated in the establishment of transcription bubble and the maintenance of the proper length of RNA-DNA hybrid; the Beta "flap", implicated in the nascent RNA binding and control of transcription termination; the secondary channel, thought to direct NTP to the catalytic center and to accept the 3' end of the nascent RNA in unproductive backtracked conformation of transcription complex.2) To clone. overexpress and purify Tag transcription factors interacting with RNAP. Genes coding for Taq RNAP sigma factors, transcription termination factors, and transcript cleavage factors will be cloned and recombinant Taq factors will be used to extend the repertoire of transcription assays for testing RNAP mutants, to obtain co-crystals with RNAP, and in collaboration with a structural group, for structure determination.The proposed work will contribute directly to our understanding of molecular basis of bacterial transcription, and will generate information necessary for development of new drugs that target bacterial RNAP. The amazing degree of RNAP conservation between bacteria and eukaryotes ensures that our results will be directly relevant to eukaryotic transcription, where the proposed analyses are not possible.