Mechanisms of transcription factor IIF function during the RNA polymerase II transcription cycle

In the yeast Saccharomyces cerevisiae, transcription frequently initiates at multiple sites ranging from 45 to 120 base pairs or greater downstream of the site of preinitiation complex assembly. Previous studies in our laboratory and others have established that RNA polymerase II (RNAPII) and the general transcription factors IIB (TFIIB) and IIF (TFIIF) play important roles in determining the positions of the mRNA 5' ends in S. cerevisiae. In this dissertation, I report the results from biochemical studies analyzing the functional alterations conferred by mutations in RNAPII and TFIIF that cause changes in transcription start site utilization. I report that the downstream shifts in start site utilization conferred by the Rpb1-R344A mutation are associated with increased abortive transcription and inability to efficiently stabilize a short RNA/DNA hybrid in the RNAPII active center. In contrast, the upstream shifts conferred by the Tfg1-E346A substitution in TFIIF are associated with both enhanced efficiency of early phosphodiester bond formation and processive elongation in vitro. These data are discussed within a proposed model for the mechanism of yeast transcription initiation in which TFIIF interaction with RNAPII modulates both early bond formation during start site utilization and conversion of RNAPII to a more processive elongating form.