Ctr9, Rtf1, and Leo1 Are Components of the Paf1/RNA Polymerase II Complex

Abstract

The Saccharomyces cerevisiae Paf1-RNA polymerase II (Pol II) complex is biochemically and functionally distinct from the Srb-mediator form of Pol II holoenzyme and is required for full expression of a subset of genes. In this work we have used tandem affinity purification tags to isolate the Paf1 complex and mass spectrometry to identify additional components. We have established that Ctr9, Rtf1, and Leo1 are factors that associate with Paf1, Cdc73, and Pol II, but not with the Srb-mediator. Deletion of either PAF1 or CTR9 leads to similar severe pleiotropic phenotypes, which are unaltered when the two mutations are combined. In contrast, we found that deletion of LEO1 or RTF1 leads to few obvious phenotypes, although mutation of RTF1 suppresses mutations in TATA-binding protein, alters transcriptional start sites, and affects elongation. Remarkably, deletion of LEO1 or RTF1 suppresses many paf1Δ phenotypes. In particular, an rtf1Δ paf1Δ double mutant grew faster, was less temperature sensitive, and was more resistant to caffeine and hydroxyurea than a paf1Δ single mutant. In addition, expression of the G₁ cyclin CLN1, reduced nearly threefold in paf1Δ, is restored to wild-type levels in the rtf1Δ paf1Δ double mutant. We suggest that lack of Paf1 results in a defective complex and a block in transcription, which is relieved by removal of Leo1 or Rtf1.

We are grateful to Taylor Washburn and Connie Phernetton for technical assistance with many aspects of these studies. We also thank Beth Amiott, Joan Betz, Kristi Penheiter, Stephanie Porter, and other members of the laboratory for advice, sharing information, and comments on the manuscript. Natalie Ahn, Katheryn Resing, David Friedman, Kim Fong, Mark Duncan, and the UCHSC Mass Spectrometry Core facility are thanked for their help with protein identification. Karen Arndt and Grant Hartzog are thanked for reagents and for freely communicating information prior to publication. David Bentley provided much useful advice and comments on the manuscript.

These studies were supported by travel funds to C.L.M. from the UCHSC Molecular Biology Program Bolie Graduate Scholarship Fund and a grant from the NIH to J.A.J. (GM38101).