STRUCTURAL & FUNCTION OF POL III TRANSCRIPTION FACTORS

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DESCRIPTION (provided by applicant) Transcription by RNA polymerase (pol) III is of fundamental importance in all eukaryotes since its products, which include 5S RNA, tRNA, U6 snRNA, RNase P RNA and 7SL RNA, are essential for protein synthesis, RNA processing, protein transport and other cellular processes. The transcription of pol III genes is tightly coupled with cell growth and is co-regulated with transcription of the large ribosomal RNAs by pol I. These transcriptional processes account for about 80% of nuclear transcription in growing cells and their coordinate regulation is thought to be important for metabolic economy and biological fitness. The majority of the proteins that comprise the pol Ill transcription machinery are conserved from yeast to humans. Thus, in almost all cases, the knowledge obtained from studies on pol III transcription in yeast is readily translated into human cells. In higher eukaryotes, pol III transcription is activated by mutations in tumor suppressor genes (p53 and RB) and by cell transformation with viral and cellular oncogenes. It is thought, therefore, that the elevated growth rate of transformed cells is dependent, in part, on their high levels of pol III transcription. The identities of the pol III transcription components that are subject to regulation under a variety of conditions and the mechanisms of their regulation are not well-defined in any system. Accordingly, the long-term objectives of this research are to identify the regulatory targets in the pol III system and determine the ways in which their function is controlled. To this end, the experiments in this application will provide a detailed biochemical understanding of a limiting step in the concerted assembly of the pol III initiation factor TFIIIB in Saccharomyces cerevisiae, namely the interaction between the tetratricopeptide repeat (TPR)- containing subunit of TFIIIC (TFIIIC131) and TFIIB-related subunit of TFIIIB (Brf1). This study will also serve as a valuable paradigm for understanding the binding specificity and function of the ubiquitous TPR motif in the assembly of multi-subunit complexes. In addition, the experiments will identify the pol III factors that respond to upstream signaling pathways and mediate transcriptional repression in yeast. These factors will be examined using biochemical and molecular genetic methods to elucidate the molecular mechanisms of their regulation. The hypotheses developed in course of these studies will be explicitly tested by a combination of in vivo and in vitro assays.