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Molecular and Cellular Biology, December 2004, p. 10975-10985, Vol. 24, No. 24
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.24.10975-10985.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Amino Acid Substitutions in Yeast TFIIF Confer Upstream Shifts in Transcription Initiation and Altered Interaction with RNA Polymerase II

Mohamed A. Ghazy,{dagger} Seth A. Brodie,{dagger} Michelle L. Ammerman, Lynn M. Ziegler, and Alfred S. Ponticelli*

Department of Biochemistry, School of Medicine and Biomedical Sciences, State University of New York, Buffalo, New York

Received 13 August 2004/ Returned for modification 13 September 2004/ Accepted 13 September 2004

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.

* Corresponding author. Mailing address: Department of Biochemistry, School of Medicine and Biomedical Sciences, State University of New York, Buffalo, NY 14214-3000. Phone: (716) 829-2473. Fax: (716) 829-2725. E-mail: asp{at}buffalo.edu.

{dagger} M.A.G. and S.A.B. contributed equally to this work.

Molecular and Cellular Biology, December 2004, p. 10975-10985, Vol. 24, No. 24
0022-538X/04/$08.00+0     DOI: 10.1128/MCB.24.24.10975-10985.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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