The DNA-dependent RNA polymerase play a central role in gene expression.
The aim of the proposed research is to produce a more precise genetic and biochemical description of the yeast RNA polymerase II. It is proposed that a combination of genetic and biochemical approaches are used to A) define the genuine components of RNA polymerase II, B) determine which subunits contribute to RNA catalysis and to interactions with promoters and transcription control factors, and C) identify transcription factors that interact directly with RNA polymerase II. To accomplish these goals, the specific aims of the experiments outlined in this proposal are: 1) to isolate and confirm the identity of the genes that specify each of the yeast RNA polymerase II subunits, by probing a recombinant DNA expression library with antibodies and by further characterization of the isolated genes. 2) to construct yeast mutants with lesions at defined RNA polymerase gene loci, resulting in conditional phenotypes, by using isolated subunit DNA sequences mutagenized in vitro to replace the wild-type gene. The conditional mutants constructed through this procedure will be used in an effort to: A) identify genuine RNA polymerase II subunits through biochemical analysis, B) identify subunits that interact with specific regulatory factors and examine the possibility that there exist multiple forms of RNA polymerase II, by analyzing the effects of the mutant alleles on the relative ability to express specific genes. 3) to isolated analyze and clone extragenic suppressors of the conditional RNA polymerase mutants in an effort to reveal RNA polymerase subunit interactions, previously undetected subunits and factors that interact with the enzyme. 4) to investigate the function of the carboxyl-terminal repeat of the large subunit of RNA polymerase II by constructing deletions in the repeat and assessing the effect on various steps in transcription. 5) to identify and isolate mutants in TATA box binding protein(s) by selecting for suppressors of a defective TATA box. Suppressing genes and their wildtype counterparts will be cloned to further study the putative protein(s). The health relatedness of this project derives from its contribution to the understanding of the basic molecular mechanisms which control gene expression.
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