The yeast Paf1 complex, minimally composed of Paf1, Ctr9, Cdc73, Rtf1, and Leo1, was originally isolated in association with RNA polymerase II (Pol II). Paf1 complex components are abundant and colocalize with Pol II on chromatin at promoters and in the coding regions of actively transcribed genes. Loss of Paf1 results in severe phenotypes and reduced amounts of other Paf1 factors, with little effect on abundance or chromatin distribution of Pol II, proteins important for transcriptional elongation (Spt5, Spt16), or RNA processing (Sub2). Loss of Paf1 factors causes a reduction of Pol II Ser2 phosphorylation and shortened poly(A) tails, suggesting that the complex facilitates linkage of transcriptional and posttranscriptional events. Surprisingly, loss of Rtf1 or Cdc73, with little phenotypic consequence, results in loss of Paf1 factors from chromatin and a significant reduction in Paf1/Pol II association. Therefore, the major functions of Paf1 can be independent of actively transcribing Pol II.

The Paf1 complex (Paf1C), composed of the proteins Paf1, Ctr9, Cdc73, Rtf1, and Leo1, accompanies RNA polymerase II (pol II) from the promoter to the 3' end formation site of mRNA and snoRNA encoding genes; it is also found associated with RNA polymerase I (pol I) on rDNA. The Paf1C is found in simple and complex eukaryotes; in human cells hSki8 is also part of the complex. The Paf1C has been linked to a large and growing list of transcription related processes including: communication with transcriptional activators; recruitment and activation of histone modification factors; facilitation of elongation on chromatin templates; and the recruitment of 3' end processing factors necessary for accurate termination of transcription. Absence of, or mutations in, Paf1C factors result in alterations in gene expression that can result in misregulation of developmental programs and loss of control of cell division leading to cancer in humans. This review considers recent information that may help to resolve whether the Paf1C is primarily a "platform" on pol II that coordinates the association of many critical transcription factors, or if the complex itself plays a more direct role in one or more steps in transcription.

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 paf1⌬ phenotypes. In particular, an rtf1⌬ paf1⌬ double mutant grew faster, was less temperature sensitive, and was more resistant to caffeine and hydroxyurea than a paf1⌬ single mutant. In addition, expression of the G 1 cyclin CLN1, reduced nearly threefold in paf1⌬, is restored to wild-type levels in the rtf1⌬ paf1⌬ 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.

The products of the yeast CDC73 and PAF1 genes were originally identified as RNA polymerase II-associated proteins. Paf1p is a nuclear protein important for cell growth and transcriptional regulation of a subset of yeast genes. In this study we demonstrate that the product of CDC73 is a nuclear protein that interacts directly with purified RNA polymerase II in vitro. Deletion of CDC73 confers a temperature-sensitive phenotype. Combination of the cdc73 mutation with the more severe paf1 mutation does not result in an enhanced phenotype, indicating that the two proteins may function in the same cellular processes. To determine the relationship between Cdc73p and Paf1p and the recently described holoenzyme form of RNA polymerase II, we created yeast strains containing glutathione S-transferase ( A minimal set of transcription factors (RNA polymerase II plus TATA-binding protein [TBP], TFIIB, TFIIE, TFIIF, and TFIIH) are necessary for mRNA promoter-specific transcription initiation in vitro (for reviews, see references 7 and 12). Regulated transcription requires, in addition to these basal factors, many accessory proteins responsible for conveying regulatory signals to the general transcriptional machinery (68). There are at least two different classes of accessory factors that have been well characterized. One class includes the TBPassociated factors (TAFs) (for a review, see reference 58). In vitro reconstitution experiments strongly implicate the TAFs in the process of transcriptional activation (10). Another class of accessory factors exists in the mediator complex associated with the C-terminal repeat domain (CTD) of the largest subunit of RNA polymerase II (for a review, see reference 33). In the yeast Saccharomyces cerevisiae, the mediator can associate with RNA polymerase II and several general initiation factors to form a large protein complex termed the holoenzyme (30, 32). Most components of the holoenzyme, including the Srbps, Gal11p, Sin4p, Rgr1p, and Swi/Snfps, were originally identified by mutations that caused transcriptional alterations in yeast (24,27,34,39,43,53,64). Although mutations in some of these gene products affect the expression of only subsets of yeast genes, an analysis of temperature-sensitive mutations of SRB4 and SRB6 revealed transcription defects at all class II promoters assayed (57). Mammalian RNA polymerase II-containing complexes that include Srbp homologs, and many of the general transcription factors as well as DNA-repair factors, have recently been described (36,42).The reported complex forms of RNA polymerase II vary widely in terms of composition. In particular, some of the general initiation factors (TBP, TFIIE, and TFIIH) are present in some complexes but not others (30,32,36,42). In addition, some of the factors, including the Srbps and Gal11p, can be found in dissociable subcomplexes (30,34). Although it is probable that some of these differences reflect the different purification protocols used to isolate these extremely large complexes from widely differing cell type...