Transcription by RNA polymerase III (pol III) is of fundamental importance in all eukaryotes since its products participate in a variety of essential cellular processes including protein synthesis, RNA processing and protein transport. The long-term goal of this work is to obtain a detailed biochemical understanding of the limiting steps in pol III gene transcription. To this end, we are studying the increased transcription associated with """"""""activating"""""""" dominant and recessive mutations in the PCF1 gene of Saccharomyces cerevisiae. PCF1 encodes the tetratricopeptide repeat (TPR)-containing subunit of TFIIIC (TFIIIC131) and is responsible for recruiting the heterotrimeric initiation factor TFIIIB to the DNA upstream of the transcription start site. The recruitment of TFIIIB is achieved by direct protein-protein interactions between TFIIIC131 and the TFIIB-related factor, TFIIIB70. The dominant and the recessive PCF1 mutations are thought to facilitate different partially limiting steps in the assembly of TFIIIB by affecting distinct protein conformational changes in TFIIIC131 and/or TFIIIB70. Biochemical experiments using purified yeast factors and recombinant proteins and molecular genetic strategies are proposed to (i) identify the steps affected and the mechanisms of """"""""activation"""""""" by the dominant and recessive PCF1 mutations, (ii) define the interaction domains between TFIIIC131 and IFIIIB70 and (iii) assess the role of TPR structure/function relationships including """"""""long range"""""""" TPR-TPR interactions in TFIIIB complex assembly. The results of these studies will provide a basis for understanding how transcriptional control of pol III gene expression may be achieved during the cell cycle or in response to cell growth rate or other chemical, biological or environmental factors. In addition, the findings will establish aparadigm for considering the function and regulation of the diverse TPR family of proteins.
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