This application describes the use of a powerful genetic strategy to target mutations in transcription factors and ancillary components of the RNA polymerase 111 transcription apparatus of yeast. This genetic approach circumvents problems encountered with the biochemical purification of the transcription factors, TFIIIB and TFIIIC, and provides a direct route to their genes. The cloning of the genes for these factors and the determination of their primary structures is the initial goal of this work. Subsequent studies will identify sites of functional importance in the cloned factors. Of particular interest are sites that are engaged in protein-protein and protein-nucleic acid interactions with other components of the transcription machinery. Finally, studies are described in which the cloned transcription factor genes are used to generate antibody probes and substrates to investigate the posttramlational regulation of transcription factor activity. The long term objectives of this work are (i) to obtain a complete molecular description of the process of transcription initiation and (ii) to gain an understanding of the mechanisms responsible for regulating transcriptional activity and hence, control of target gene expression. This system serves as a model for higher eukaryotes and offers the advantages of combined genetic, molecular biological and biochemical technologies to study the primary event in gene expression, namely, the transcription of RNA. Several genetic strategies, based on a common theme, are presented: A unique tandem arrangement of tRNA nonsense suppressor genes is described in which expression of a downstream (supS1) gene is dependent upon transcription directed by the internal promoters of an upstream (sup9-e) gene. Different promoter mutations in latter prevent supS1 expression and thus provide a selection for extragenic mutations that suppress the transcriptional defect. Mutant strains have been isolated that suppress the effect of an A-block (sup9-e A19) promoter mutation. Genetic characterization, complementation analysis and a gene cloning protocol are described. In vitro transcription experiments with whole-cell extracts and fractionated transcription components from the mutant strains are proposed as a means to identify the mutant factors and study their mechanism of action. Studies on transcription factor structure- function relationships and the regulation of transcription factor activity will be conducted using these in vitro systems together with in vivo and in vitro mutagenesis strategies.
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