We are using a combination of genetics and biochemistry to elucidate properties of RNA polymerase II and other components of the eukaryotic transcription apparatus; our long term objective is a better understanding of mechanisms regulating gene expression. We plan to complete the DNa sequence of the Drosophila melanogaster RpII215 locus, a gene we previously identified and cloned which encodes the largest subunit (215 kDa) of RNA polymerase II. We also will complete our description of the structures and expression of the RNA transcript of the locus. Using transposon-mediated gene transfer experiments, we will map the RpII215C4 mutation (which renders RNA polymerase resistant to the specific inhibitor, alpha-amanitin) to a small subinterval of the locus and sequence that subinterval to identify the mutation. Other mutations in the gene will be identified analogously; these identifications should begin to define functional domains in the large subunit. To enhance our understanding of RNA polymerse II, we will clone genes for other subunits of the enzyme. To accomplish this we will screen a bacterial expression vector library using antibodies against subunits of the enzyme. We will characterize the clones we isolate to establish which subunits they represent; we will use the clones to map the new RpII loci; we will generate mutations in the new RpII loci; and we will analyze the mutant loci and the enzymes they encode. We plan to analyze both wild types and mutant polymerases in vitro using an extract that supports accurate initiation at promoter sites. We hope to identify specific roles for individual polymerase subunits in the complicated process of RNA synthesis. We also anticipate that this approach will help elucidate the nature and functions of other components necessary for proper transcription of cloned genes. Finally, we hope to identify directly other transcriptionally important components by isolating second-site suppressors of RpII mutations.
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