The work described in this application exploits the genetic and biochemical advantages offered by yeast to study a process of fundamental importance in all eukaryotes, namely transcription by RNA polymerase III (pol III). Pol III gene products participate in a myriad of essential cellular processes including protein syntheses; pre-mRNA, pre-rRNA and pre-tRNA maturation; protein transport and telomere synthesis. The expression of pol III genes must be appropriate for the norma execution of these processes and must be co-ordinated with cell growth. The long term goal of this work is to obtain a detailed biochemical understanding of how pol III genes are expressed and regulated at the transcriptional level. To this end, a combination of molecular genetic and biochemical approaches are being used to investigate the nature and function of factors that play crucial but presently poorly defined roles in pol III transcription. a unique genetic strategy has yielded mutations in four genes (PCF1 to PCF4), each of which increases transcription by pol III. The PCF1-1 mutation influences the rate of recruitment of transcription factor (TF) IIIB to DNA. This effect will be examined by time-resolved footprinting. Structure-function studies of PCF1 are also proposed in which new dominant alleles and conditional mutations will be isolated. The latter will be used to clone genes for interacting polypeptides by multi-copy suppression. Studies on PCF1-1 suggest that the binding of TFIIIB to DNA is accompanied by a posttranslational event which activates a subunit of this factor, TFIIIB90. Direct evidence that TFIIIB90 is posttranslationally modified will be sought by in vivo labeling experiments following the purification and cloning of this factor. Our present transcriptional model suggests that PCF2-1 may encode the catalytic activity responsible for TFIIIB90 activation. The nature and function of PCF2 will be determined by gene sequencing and transcription studies. The PCF4-1 mutation identifies a polymerase specificity factor, TFIIIB70, that is stoichiometrically limiting transcription. Experiments are proposed to examine the effect of over-expressing this protein in yeast and to study its interactions with other components of the transcription machinery. Finally, a series of biochemical and molecular experiments are outlined to test the hypothesis that PCF3, a negative regulator of pol III transcription, mediates growth control over this process.
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