Yeast RNA Polymerase II Lacking the Rpb9 Subunit Is Impaired for Interaction with Transcription Factor IIF

Lynn M Ziegler,Denys A Khaperskyy,Michelle L Ammerman,Alfred S Ponticelli

doi:10.1074/jbc.m309656200

Lynn M Ziegler, Denys A Khaperskyy + Show 2 more

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https://doi.org/10.1074/jbc.m309656200

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Abstract

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.

Highlights

The initiation of mRNA synthesis by eukaryotic RNA polymerase II (RNAPII)1 is a critical step in the regulation of eukaryotic gene expression
Transcription assays were performed using plasmid templates containing either the S. cerevisiae ADH1 or HIS4 promoters; purified recombinant factors (TBP, transcription factors IIB (TFIIB), TFIIE, and TFIIF); and wild-type RNAPII, the ⌬Rpb9 RNAPII, and TFIIH purified from yeast extracts
We have investigated the molecular basis underlying the alterations in the positions of mRNA 5Ј-ends that are conferred both in vivo and in vitro by an S. cerevisiae RNA polymerase II mutant lacking the Rpb9 subunit

Summary

Introduction

The initiation of mRNA synthesis by eukaryotic RNA polymerase II (RNAPII)1 is a critical step in the regulation of eukaryotic gene expression. The results demonstrated that upstream shifts in the positions of mRNA 5؅-ends could be conferred by the ⌬Rpb9 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 ⌬Rpb9 polymerase and TFIIF.

Results

Conclusion