Ctr9, Rtf1, and Leo1 are components of the Paf1/RNA polymerase II complex.

RNA polymerase II (Pol II) requires both general transcription factors (GTFs) to form a stable initiation complex at promoters (reviewed in references 6, 24, and 60) and mediators and coactivators to communicate signals from transcriptional activators and repressors (reviewed in reference 36). Large, apparently preassembled forms of Pol II including many of these accessory factors may be the actual initiating forms of the enzyme in vivo. However, recent studies have demonstrated that mediator complexes may also function in the absence of Pol II (7, 3a, 40). The best characterized of these holoenzyme forms of Pol II is the Srb-mediator complex from Saccharomyces cerevisiae (reviewed in references 34 and 35). In addition to core Pol II, this complex contains the mediator, a collection of about 20 proteins including the Srb proteins, and the GTFs transcription factor IIB (TFIIB), TFIIF, TFIIE, and TFIIH. Complexes similar to the yeast Srb-mediator have also been described in multicellular organisms (reviewed in reference 13). Our laboratory has isolated and characterized a Pol II complex from yeast containing Paf1, Cdc73, Hpr1, and Ccr4 that is biochemically distinct from the Srb-mediator complex (4, 47, 57). The Paf1 complex contains TFIIF and TFIIB and the coactivators Gal11 and Sin4 that are also present in the Srb-mediator complex. However, the Srb and Med proteins are not found in the Paf1 complex, and Paf1, Cdc73, Hpr1, and Ccr4 are not found with the proteins of the Srb-mediator complex. Although genes encoding Paf1 complex components are not essential, mutations in these genes exhibit a variety of phenotypes indicative of defects in gene expression (4, 47; J. L. Betz, M. Chang, T. M. Washburn, S. E. Porter, and J. A. Jaehning, submitted for publication). Consistent with these phenotypes, mutation of Paf1 complex genes results in changes in abundance of a small but significant subset of yeast transcripts (4, 47; M. Chang, J. Fostel, and J. A. Jaehning, unpublished data). In contrast, many of the genes encoding Srb and Med proteins are essential (reviewed in reference 34), and loss of at least one of the essential Srb proteins (Srb4) results in diminished expression of most, if not all, yeast genes (18). Transcripts differentially expressed in strains lacking Paf1 include cell wall biosynthetic genes (4) and many cell cycle-regulated genes (S. E. Porter, R. M. Washburn, M. Chang, and J. A. Jaehning, submitted for publication). Loss of Paf1, Cdc73, or Ccr4 results in cell wall integrity defects, and loss of Paf1, Cdc73, or Hpr1 is correlated with elevated rates of recombination between direct repeats (4). The fact that these two otherwise unrelated phenotypes are also observed for mutations in genes in the protein kinase C-mitogen-activated protein kinase signaling pathway led us to establish that the Paf1 complex plays an important role in the transmission of signals from Pkc1 to downstream target genes (4). Although Paf1 and Cdc73 have not been identified in other complexes, both Hpr1 and Ccr4 are present in other non-Pol II-containing complexes. Hpr1 is part of the Hpr1/THO complex that influences transcription and recombination (5), and Ccr4 is part of the Ccr4/Caf1/NOT complex that functions in both transcript synthesis and turnover (27, 28, 52). Recently Koch and coworkers identified Paf1 and Cdc73 as factors associated with Ctr9, a protein required for full expression of the G1 cyclin CLN2 (22). Loss of Paf1 or Ctr9 leads to similar severe pleiotropic phenotypes, and loss of both genes does not result in an enhanced phenotype. These genetic and biochemical observations are consistent with the idea that Ctr9 and Paf1 function in the same pathways. To further characterize the Paf1 complex, and to determine whether Ctr9 is also one of the Pol II-associated proteins in this complex, we have used the tandem affinity purification (TAP) (43) system to isolate the Paf1 complex. We have confirmed the presence of Ctr9 in the Pol II complex, and we have identified new components of the Paf1 complex, including Rtf1 and Leo1. The absence of these newly identified factors from the Srb-mediator complex further establishes the biochemical differences between these two forms of Pol II. Our observation that loss of Rtf1 or Leo1 suppresses many of the pleiotropic phenotypes caused by loss of Paf1 or Ctr9 has led us to a new model of the role of the Paf1 complex in transcription of yeast genes.