Functions of Saccharomyces cerevisiae TFIIF during Transcription Start Site Utilization

Abstract

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...

Inactivated RNA Polymerase II Open Complexes Can Be Reactivated with TFIIE

“…We presume, based on our recent work with conventional templates (22), that bubble template PICs assembled with P-TFIIF do not retain TFIIF. Our results with bubble templates are therefore in general agreement with the earlier work (28,39). Why should the presence of TFIIF within PICs have very different effects on doublestranded versus bubble templates?…”

“…It was reported earlier (28,39) that transcript initiation and promoter clearance are stimulated by TFIIF. These experiments relied on premelted templates in order to study transcription in the presence or absence of TFIIF.…”

Transcriptional Regulation inSaccharomyces cerevisiae: Transcription Factor Regulation and Function, Mechanisms of Initiation, and Roles of Activators and Coactivators

“…Consistent with this, DNA in vivo at the GAL1 and GAL10 promoters is unwound from 20 bp downstream of TATA (the approximate site of initial strand unwinding for metazoan Pol II) through the transcription start 90 bp distant from the TATA (Giardina and Lis 1993). This scanning mechanism does not involve transcription of the DNA between the TATA and initiation site (Khaperskyy et al 2008). Since the cost of disrupting a DNA base pair is 2 kcal/mol, there is a significant energetic cost of unwinding 10-70 bp.…”

“…These mutations act as though they decrease the efficiency of initiator recognition. In contrast, mutations in the TFIIF dimerization domain or in two subunits of Pol II that interact with TFIIF, Rpb2, and Rpb9, start transcription closer to TATA than in wild-type cells and behave as though they have relaxed specificity for initiator recognition (Khaperskyy et al 2008). The study of this mechanistic step in S. cerevisiae will undoubtedly reveal important details about start site selection in other eukaryotes.…”

Conserved architecture of the core RNA polymerase II initiation complex

“…The arm forms 19 crosslinks in the cleft, consistent with detection of the arm in the human PIC by EM 7 . A mutation at the point where the arm extends from the dimerization module leads to shifts in the transcription start site 24 .…”