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.
Ghazy
Mohamed A
MA
Department of Biochemistry, School of Medicine and Biomedical Sciences, State University of New York, Buffalo, NY 14214-3000, USA.
Brodie
Seth A
SA
Ammerman
Michelle L
ML
Ziegler
Lynn M
LM
Ponticelli
Alfred S
AS
D000409:Alanine / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics*; D024202:Electrophoretic Mobility Shift Assay; D005816:Genetic Complementation Test; D015151:Immunoblotting; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D017434:Protein Structure, Tertiary; D012319:RNA Polymerase II / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D019473:Transcription Factors, TFII / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic
D019473:Transcription Factors, TFII; D012319:RNA Polymerase II; C056604:transcription factor TFIIF; D000409:Alanine