The major goal of the work proposed in this application is the investigation of the structure-function relationships of the Beta and Beta' subunits of core RNA polymerase of E. coli by a genetic, physiological and biochemical analysis of mutants. Five different classes of RNA polymerase mutants will be chosen for intensive analysis: rifampicin resistant mutants, streptolydigin resitant mutants, mutants with altered sigma interaction, mutants with altered DNA binding and mutants that exhibit a dominant lethal phenotype. We will use deletion mapping to locate precisely the mutations within rpoB (coding for Beta) or rpoC (coding for Beta'). In vivo analysis of regulation of gene activity and in vitro analysis of DNA binding and transcription will enable us to assess the nature of the functional alteration in the mutant RNA polymerase. Based on such studies, we will be able to determine whether mutations resulting in the same or similar functional alterations are clustered on the genetic map. DNA sequencing of clustered mutations will enable us to correlate functional defect with changes in primary and secondary structure of the protein. Finally, second site revertants of some mutant classes will be used to identify factors which interact with core polymerase.
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