This study will characterize an important protein, TFIIIC, that is essential for the expression of a large class of genes in eucaryotic cells which are transcribed by RNA polymerase III. Specifically, one of the first critical steps in the transcription process will be examined: the direct interaction of TFIIIC with the DNA template. To this end, a partially purified fraction containing the yeast Saccharomyces cerevisiae TFIIIC will be used to determine the specific nucleotides within the two internal promoter regions of the tRNA gene required for its binding. Since the yeast system also exhibits a dependence for 5'flanking sequence for efficient tRNA gene transcription, the additional contribution of these sequences on TFIIIC binding will also be assessed. These studies will use a variety of mutant templates previously derived from two tRNA genes. Nuclease-protection assays will qualitalively determine the site of TFIIIC binding to these templates. Gel retardation analysis of TFIIIC-DNA complexes will quantitate how these mutations affect the affinity of TFIIIC for these templates. These studies will also examine the structure of the TFIIIC polypeptide responsible for this specific interaction. Recently, using a protein blotting procedure, a 150,000 dalton molecular weight protein has been identified which specifically interacts with tRNA genes and constitutes the DNA binding function of TFIIIC. This study will elucidate whether this protein contains functional DNA binding domains which are responsible for its interaction with one or both of the internal promoter regions. Further purification of TFIIIC by conventional and affinity chromatography methods will allow for the generation of monospecific antibodies and protein sequence information. Yeast genomic libraries will be screened using immunological and hybridization methods for isolating the gene encoding the TFIIIC protein. Overproduction of the TFIIIC polypeptide will be accomplished using a yeast expression system. Potential DNA- binding and protein-binding domains on this protein will be delineated by assaying its ability to bind specifically to tRNA genes and to reconstitute tRNA transcription in vitro with TFIIIB and RNA polymerase III. Future studies will involve mutagenesis of the TFIIIC gene and the production of mutant TFIIIC proteins. These studies will determine the structure function relationship of the TFIIIC polypeptide and delineate ordered interactions which define TFIIIC's role in the mechanism of RNA polymerase III transcription.
