The first aim asks how Mot1, a TBP associated protein, functions to mediate Leu3-dependent repression. Initial experiments test for a direct interaction between Leu3 and Mot1 (either alone or in TBP complex). To study the Mot1-containing TBP complex, Dr. Jaehning proposes to fully reconstitute an in vitro transcription system with defined components. This system will be used to study the mechanism of repression by Mot1 (e.g. template commitment, polymerase or general factor recruitment and promoter clearance).
Aim 2 focuses on the RNA polymerase Associated Proteins (RAPs), specifically Paf1 and Cdc73. Dr. Jaehning plans to isolate RNA polymerase complexes that are contain either Paf1 or SRBs from strains containing tagged versions of RAPs, SRBs or TFIIF. The relative frequency of the various types of complexes will be monitored during growing stages and the cell cycle. To study the function of the Paf1-type complex, Paf1 responsive promoters will be identified by differential display. A possible cell cycle role for Paf1 and its regulation by phosphorylation will be pursued. The Gal1 or Gal10 promoters will be used initially to monitor the mechanism of Paf1 action. Initiation vs. promoter clearance vs. transcript stability will be monitored.
The third aim addresses the mechanism of factor-specific activation using the regulated activators, Leu3 and Gal4. A combination of purified general factors and partially purified fractions will be used in vitro transcription assays. If fractions are found that are specifically required for one of the activators, components will be screened with antibodies to known factors. Direct interaction assays between activators and specific components will be performed. Finally, factors responsible for Gal4 phosphorylation will be investigated by testing the candidate Gal3 and possible activating fractions.
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