Cdc73p and Paf1p Are Found in a Novel RNA Polymerase II-Containing Complex Distinct from the Srbp-Containing Holoenzyme

Abstract

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...

Global Screening of Genes Essential for Growth in High-Pressure and Cold Environments: Searching for Basic Adaptive Strategies Using a Yeast Deletion Library

“…The slm3D mutation is known to be synthetically lethal with the cdc73D mutation (Tong et al 2004). CDC73 encodes a constituent of the Paf1 complex with RNA polymerase II (Shi et al 1997;Krogan et al 2003). Interestingly, the cdc73D mutant was also unable to grow under high pressure and low temperature (see below).…”

Nuclear Localization of the Parafibromin Tumor Suppressor Protein Implicated in the Hyperparathyroidism-Jaw Tumor Syndrome Enhances Its Proapoptotic Function

“…2A-C). The latter fragment of parafibromin contains the region from residues 233 to 525 homologous to cdc73p, the yeast RNA polymerase II accessory factor component of the Paf1 complex (20,21). Because yeast cdc73p and the other yeast Paf1 complex components are nuclear proteins (22), and because human parafibromin (hCdc73) has been identified as a component of a human PAF1 complex (12)(13)(14), it was expected that GFP-HRPT2-C would exhibit nuclear localization like the full-length parafibromin fusion (GFP-HRPT2-A).…”

Elongation by RNA polymerase II: the short and long of it

“…A description of the role of FCP1 in the CTD phosphorylation cycle is provided below. Further experimentation identified that TFIIF associates with multiple elongation factors, including the Spt5 subunit of DSIF , and components of the PAF complex (Shi et al 1997). TFIIF also appears to influence the TFIIS cleavage factor (see below) (Elmendorf et al 2001;Zhang et al 2003a).…”

“…Paf1 is found in a complex with four additional subunits, Ctr9, Cdc73, Rtf1, and Leo1 (Shi et al 1996(Shi et al , 1997Krogan et al 2002b;Mueller and Jaehning 2002;Squazzo et al 2002). Genetic studies of PAF subunits revealed a wide range of phenotypes, including transcript elongation phenotypes (Costa and Arndt 2000;Mueller and Jaehning 2002;Squazzo et al 2002;Mueller et al 2004).…”