The broad long-term objectives of this proposal are to (10 elucidate at a molecular level, the pathway of transcription initiation by RNA polymerase II (RNAIPII) and (2) determine how the steps in this pathway are affected by gene-specific regulatory proteins.
The specific aims are to identify and purify the RNAPII general transcription factors for S. cerevisiae, to clone and analyze the gene encoding the TATA binding factor. TFIID, and to analyze at a biochemical level how this protein interacts with DNA as well as other components of the transcriptional machinery and gene-specific regulatory proteins. Understanding the fundamental process of gene control will provide a basis for understanding complex patterns of gene expression regulating development and the growth of animal cells. The yeast general transcription factors TFIIB,E will be identified by reconstitution of a homologous yeast in vitro transcription system. These factors, as well as the yeast general factor TFIIA will be purified. Using the already determined amino acid sequence of TFIID, the gene encoding this factor will be cloned and characterized. The yeast TFIID gene will be used to create TRIIF mutations in yeast to study its function, determine whether it is part of a multigene family and to study the regulation of its expression. Genes encoding factors which directly interact with TFIID will be identified by genetic screening and biochemical analysis. The TFIID gene will be mutagenized in vitro and the effect of these mutations will be assessed both in vivo and in vitro to define the functional domains of the protein. Using chemical premodification and mutagenesis of the TFIID DNA binding sites, the critical protein-DNA interactions will be determined. Finally, the assembly of the general factors with RNAPII will be studied in vitro using a native gel assay and DNAsel protection. The kinetics of general factor assembly will be examined in both the presence and absence of transcriptional regulatory proteins to identify regulated steps in the pathway of transcription initiation.
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