Previous studies have shown that substitutions in the Tfg1 or Tfg2 subunits of Saccharomyces cerevisiae transcription factor IIF (TFIIF) can cause upstream shifts in start site utilization, resulting in initiation patterns that more closely resemble those of higher eukaryotes. In this study, we report the results from multiple biochemical assays analyzing the activities of wild-type yeast TFIIF and the TFIIF Tfg1 mutant containing the E346A substitution (Tfg1-E346A). We demonstrate that TFIIF stimulates formation of the first two phosphodiester bonds and dramatically stabilizes a short RNA-DNA hybrid in the RNA polymerase II (RNAPII) active center and, importantly, that the Tfg1-E346A substitution coordinately enhances early bond formation and the processivity of early elongation in vitro. These results are discussed within a proposed model for the role of yeast TFIIF in modulating conformational changes in the RNAPII active center during initiation and early elongation.Transcription of eukaryotic protein-encoding genes is a multistep process involving the concerted actions of RNA polymerase II (RNAPII) and a host of auxiliary factors that include the general transcription factors (TF)IIB, TFIID, TFIIE, TFIIF, and TFIIH (17,18,32). In higher eukaryotes, the architecture of the preinitiation complex (PIC) is believed to be the main determinant for the site of transcription initiation. For promoters that contain a TATA element, transcription typically initiates at a single site within the region that initially undergoes ATP-dependent DNA strand separation approximately 30 base pairs downstream of the TATA element. In striking contrast, mRNA 5Ј ends in the yeast Saccharomyces cerevisiae frequently map to multiple sites within an extended window ranging from 45 to 200 base pairs downstream of the TATA element (9, 42). Although the mechanistic basis for this difference remains unknown, results from both in vivo and in vitro studies suggest that both the overall architectures and the relative locations of promoter melting for human and S. cerevisiae PICs are similar (15,31).Previous studies in our laboratory and by others have established that RNAPII and the general factors TFIIB and TFIIF play critical roles in determining the positions of the mRNA 5Ј ends in S. cerevisiae. Mutations in the highly conserved B finger domain of TFIIB, or in the switch 2 region of the RNAPII Rpb1 subunit, can cause downstream shifts in the positions of mRNA 5Ј ends (1,10,33,35,36,50). Conversely, mutations in the Rpb2 or the Rpb9 polymerase subunit (5, 21, 43, 52) or in the TFIIF Tfg1 or Tfg2 subunit (14) can confer upstream shifts in start site utilization, resulting in initiation patterns that more closely resemble those of higher eukaryotes.To date, relatively few studies have been conducted to analyze the functions of S. cerevisiae TFIIF, in contrast to the numerous reports that have demonstrated activities for mammalian TFIIF during multiple steps of the transcription cycle. Mammalian TFIIF has been shown to assist in the recruitme...

RNA polymerase II (RNAPII) is responsible for the synthesis of mRNA from eukaryotic protein-encoding genes. In this study, sitedirected mutagenesis was employed to probe the function of residues within the Saccharomyces cerevisiae RNAPII active center in the mechanism of transcription start site utilization. We report here the identification of two mutations in the switch 2 region, rpb1-K332A and rpb1-R344A, which conferred conditional growth properties and downstream shifts in start site utilization. Analyses of double mutant strains demonstrated functional interactions between these switch 2 mutations and a mutation in the largest subunit of transcription factor IIF (TFIIF) that confers upstream shifts in start site usage. Importantly, biochemical analyses demonstrated that purified Rpb1-R344A mutant polymerase exhibited impaired ability to stabilize a short RNA-DNA hybrid in the active center, an increased frequency of abortive transcription in runoff assays, and both a downstream shift and increased abortive initiation in reconstituted transcription assays. These results provide evidence for a role of switch 2 during start site utilization and indicate that RNA-DNA hybrid stability at the 3-end of the transcript is a determinant in this process. We discuss these results within the context of a proposed model regarding the concerted roles of RNA-PII, TFIIB, and TFIIF during mRNA 5-end formation in S. cerevisiae.The synthesis of mRNA from protein-encoding (class II) genes in eukaryotic organisms is a regulated multistep process that involves the concerted action of RNA polymerase II (RNAPII) 2 and a host of auxiliary transcription factors (1, 2). In higher eukaryotes, transcription initiation typically occurs ϳ30 base pairs downstream of a TATA element. In contrast, mRNA 5Ј-ends in the yeast Saccharomyces cerevisiae frequently map to multiple sites within a window ranging from 40 to 120 base pairs or greater downstream of a TATA element (3,4). Although the precise mechanism by which RNAPII recognizes and productively utilizes a potential start site remains to be elucidated, previous studies have firmly established that RNAPII and the general transcription factors IIB (TFIIB) and IIF (TFIIF) play important roles in determining the position of mRNA 5Ј-ends in S. cerevisiae. Numerous genetic and biochemical studies of S. cerevisiae TFIIB conducted in our laboratory and by others have shown that amino acid substitutions in a highly conserved N-terminal region of TFIIB can alter the position of mRNA 5Ј-ends both in vivo and in vitro (5-10). Specifically, these TFIIB mutations confer a downstream shift in start site utilization, i.e. diminished utilization of sites located proximal to the TATA element with accompanying enhanced utilization of sites located further downstream. Interestingly, the structural determination of an S. cerevisiae TFIIB-RNAPII co-crystal has revealed that this N-terminal region of TFIIB adopts a finger-like structure that projects into the RNA exit channel of the polymerase and is positioned...