Amino acid substitutions in yeast TFIIF confer upstream shifts in transcription initiation and altered interaction with RNA polymerase II.

Transcription factor IIF (TFIIF) is required for transcription of protein-encoding genes by eukaryotic RNA polymerase II. In contrast to numerous studies establishing a role for higher eukaryotic TFIIF in multiple steps of the transcription cycle, relatively little has been reported regarding the functions of TFIIF in the yeast Saccharomyces cerevisiae. In this study, site-directed mutagenesis, plasmid shuffle complementation assays, and primer extension analyses were employed to probe the functional domains of the S. cerevisiae TFIIF subunits Tfg1 and Tfg2. Analyses of 35 Tfg1 alanine substitution mutants and 19 Tfg2 substitution mutants identified 5 mutants exhibiting altered properties in vivo. Primer extension analyses revealed that the conditional growth properties exhibited by the tfg1-E346A, tfg1-W350A, and tfg2-L59K mutants were associated with pronounced upstream shifts in transcription initiation in vivo. Analyses of double mutant strains demonstrated functional interactions between the Tfg1 mutations and mutations in Tfg2, TFIIB, and RNA polymerase II. Importantly, biochemical results demonstrated an altered interaction between mutant TFIIF protein and RNA polymerase II. These results provide direct evidence for the involvement of S. cerevisiae TFIIF in the mechanism of transcription start site utilization and support the view that a TFIIF-RNA polymerase II interaction is a determinant in this process.

Yeast RNA polymerase II lacking the Rpb9 subunit is impaired for interaction with transcription factor IIF.

Previous studies have shown that transcription factors IIB (TFIIB), IIF (TFIIF), and RNA polymerase II (RNAPII) play important roles in determining the position of mRNA 5'-ends in the yeast Saccharomyces cerevisiae. Yeast strains containing a deletion of the small, nonessential Rpb9 subunit of RNAPII exhibit an upstream shift in the positions of mRNA 5'-ends, whereas mutation of the large subunit of yeast TFIIF (Tfg1) can suppress downstream shifts that are conferred by mutations in TFIIB. In this study, we report an approach for the production of functional recombinant yeast holo-TFIIF (Tfg1-Tfg2 complex) and use of the recombinant protein in both reconstituted transcription assays and gel mobility shifts in order to investigate the biochemical alterations associated with the deltaRpb9 polymerase. The results demonstrated that upstream shifts in the positions of mRNA 5'-ends could be conferred by the deltaRpb9 RNAPII in transcription reactions reconstituted with highly purified yeast general transcription factors and, importantly, that these shifts are associated with an impaired interaction between the DeltaRpb9 polymerase and TFIIF. Potential mechanisms by which an altered interaction between the DeltaRpb9 RNAPII and TFIIF confers an upstream shift in the positions of mRNA 5'-ends are discussed.