Functional interaction between Ssu72 and the Rpb2 subunit of RNA polymerase II in Saccharomyces cerevisiae.
SSU72 is an essential gene encoding a phylogenetically conserved protein of unknown function that interacts with the general transcription factor TFIIB. A recessive ssu72-1 allele was identified as a synthetic enhancer of a TFIIB (sua7-1) defect, resulting in a heat-sensitive (Ts(-)) phenotype and a dramatic downstream shift in transcription start site selection. Here we describe a new allele, ssu72-2, that confers a Ts(-) phenotype in a SUA7 wild-type background. In an effort to further define Ssu72, we isolated suppressors of the ssu72-2 mutation. One suppressor is allelic to RPB2, the gene encoding the second-largest subunit of RNA polymerase II (RNAP II). Sequence analysis of the rpb2-100 suppressor defined a cysteine replacement of the phylogenetically invariant arginine residue at position 512 (R512C), located within homology block D of Rpb2. The ssu72-2 and rpb2-100 mutations adversely affected noninduced gene expression, with no apparent effects on activated transcription in vivo. Although isolated as a suppressor of the ssu72-2 Ts(-) defect, rpb2-100 enhanced the transcriptional defects associated with ssu72-2. The Ssu72 protein interacts directly with purified RNAP II in a coimmunoprecipitation assay, suggesting that the genetic interactions between ssu72-2 and rpb2-100 are a consequence of physical interactions. These results define Ssu72 as a highly conserved factor that physically and functionally interacts with the RNAP II core machinery during transcription initiation.
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Department of Biochemistry, Division of Nucleic Acids Enzymology, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.
Hampsey
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D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000235:genetics* / Q000502:physiology; D017124:Conserved Sequence; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D010749:Phosphoprotein Phosphatases; D010802:Phylogeny; D021122:Protein Subunits; D012319:RNA Polymerase II / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D013489:Suppression, Genetic; D035581:Transcription Factor TFIIB; D014157:Transcription Factors / Q000235:genetics; D014158:Transcription, Genetic; D039221:mRNA Cleavage and Polyadenylation Factors
D002352:Carrier Proteins; D005656:Fungal Proteins; D021122:Protein Subunits; C416934:SSU72 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D035581:Transcription Factor TFIIB; D014157:Transcription Factors; D039221:mRNA Cleavage and Polyadenylation Factors; D012319:RNA Polymerase II; D010749:Phosphoprotein Phosphatases