Functional interaction between Ssu72 and the Rpb2 subunit of RNA polymerase II in Saccharomyces cerevisiae.

Eukaryotic RNA polymerase II (RNAP II) is a multisubunit enzyme that is responsible for transcription of all protein-encoding genes. Saccharomyces cerevisiae RNAP II is a 12-subunit complex encoded by the genes RPB1 to RPB12 (reviewed in references 2, 53, and 55). The two largest subunits, Rpb1 and Rpb2, are homologous to the β′ and β subunits of bacterial RNA polymerase (RNAP), respectively (23, 49). The yeast counterpart of the bacterial α subunit appears to be shared between the Rpb3 and Rpb11 subunits (26, 57). Accordingly, Rpb1, Rpb2, Rpb3, and Rpb11 comprise the functional equivalent of the bacterial α2ββ′ core RNAP. The functions of the remaining RNAP II subunits are less clear, although five subunits, Rpb5, Rpb6, Rpb8, Rpb10, and Rpb12, are shared among all three forms of RNAP and therefore function in transcription of all genes. With the exception of Rpb4 and Rpb9, all subunits are essential for cell viability. Rpb4 forms a subcomplex with Rpb7 that is required for promoter-specific initiation but is dispensable for elongation (13). RNAP II is unable to initiate promoter-specific transcription on its own. Promoter recognition requires the general transcription factors (GTFs), which include the TATA binding protein (TBP), TFIIB, TFIIE, TFIIF, and TFIIH (reviewed in references 17 and 33). TBP nucleates assembly of a transcription preinitiation complex by binding the TATA element and inducing a sharp bend in the DNA template. TFIIB binds the TATA-TBP complex and is responsible for defining the polarity of transcription by binding asymmetrically to the BRE element, located immediately upstream of the TATA box (28, 50). RNAP II, in association with TFIIF, binds the TATA-TBP-TFIIB ternary complex, followed by TFIIE and TFIIH. TFIIH is a multisubunit complex with catalytic activities responsible for phosphorylation of the carboxy-terminal repeat domain of the Rpb1 subunit of RNAP II, promoter melting and promoter clearance (25). The GTFs were discovered based on their requirement for accurate initiation in vitro (29, 52) and are highly conserved among eukaryotic organisms (reviewed in references 17, 30, 33, and 38). Furthermore, the TBP and TFIIB requirements for transcription predate the divergence of Eukarya and Archaea (reviewed in reference 5). Nonetheless, the RNAP II requirement for GTFs is not universal but is instead dictated by promoter sequence and architecture (16, 35, 51). Indeed, genome-wide expression analysis revealed that most components of the RNAP II transcriptional machinery are dispensable for expression of subsets of genes (21). Conversely, since only a limited number of promoters have been analyzed in vitro, it is conceivable that additional GTFs might be identified based on their requirement for accurate initiation from specific promoters. Other factors might be identified based on their ability to stimulate activator-independent transcription. For example, the yeast Tsp1 (Sub1) protein was identified as a positive effector of in vitro transcription in the absence of a sequence-specific activator (20). Tsp1 interacts physically and genetically with TFIIB and is homologous to the human transcriptional cofactor PC4 (20, 27). Additional basal factors are likely to be identified, either by their requirement for promoter-specific initiation, by their ability to stimulate activator-independent transcription, or by genetic interactions with GTFs or RNAP II subunits. The yeast gene encoding TFIIB (SUA7) was identified as an effector of transcriptional accuracy (36). Replacements at either of two phylogenetically invariant residues, glutamate-62 (E62) or arginine-78 (R78), caused a marked start site shift downstream of the normal site at the CYC1 and ADH1 promoters (37). Nearly identical effects on transcriptional accuracy are conferred by the sua8 alleles of RPB1, suggesting a functional interaction between TFIIB and Rpb1 during start site selection (6). In an effort to identify other factors that affect initiation, we isolated suppressors of the sua7-1 (E62K) mutation. The ssu71 and ssu73 suppressors encode altered forms of the largest subunit of TFIIF (Tfg1) and the Rpb9 subunit of RNAPII, respectively (46, 47). In contrast, ssu72 was identified as an enhancer of the TFIIB E62K defect: the ssu72-1 allele confers a heat-sensitive (Ts−) growth defect and a dramatic downstream start site shift, with both effects being dependent upon the sua7-1 allele (48). The SSU72 gene is essential for cell viability and encodes a novel protein of undefined function (48). To further characterize Ssu72, we generated a new ssu72 allele that confers a tight Ts− phenotype, independent of a TFIIB defect. We have taken advantage of the ssu72 Ts− phenotype to isolate a new rpb2 mutation encoding an altered from of the Rpb2 subunit of RNAP II. The results presented here demonstrate that Ssu72 is a transcription factor that interacts with the core RNAP II machinery both in vivo and in vitro.