Transcription and processing of pre-mRNA are coupled events. By using a combination of biochemical, molecular, and genetic methods, we have found that the phylogenetically conserved transcription factor Ssu72 is a component of the cleavage/polyadenylation factor (CPF) of Saccharomyces cerevisiae. Our results demonstrate that Ssu72 is required for 3 end cleavage of pre-mRNA but is dispensable for poly(A) addition and RNAP II termination. The in vitro cleavage defect caused by depletion of Ssu72 from cells can be rescued by addition of recombinant Ssu72. Ssu72 interacts physically and genetically with the Pta1 subunit of CPF. Overexpression of PTA1 causes synthetic lethality in an ssu72-3 mutant. Moreover, Sub1, which has been implicated in transcription initiation and termination, also interacts with Pta1, and overexpression of SUB1 suppresses the growth and processing defect of a pta1 mutation. Physical interactions of Ssu72 and Sub1 with Pta1 are mutually exclusive. Based on the interactions of Ssu72 and Sub1 with both the Pta1 of CPF and the TFIIB component of the initiation complex, we present a model describing how these novel connections between the transcription and 3 end processing machineries might facilitate transitions in the RNAP II transcription cycle.
The establishment of DNA synthesis during the S phase is a multistep process that occurs in several stages beginning in late mitosis. The first step is the formation of a large prereplicative complex (pre-RC) at individual replication origins and occurs during exit from mitosis and entry into G1 phase. To better understand the genetic requirements for pre-RC formation, we selected chromosomal suppressors of a temperature-sensitive cdc6-4 mutant defective for pre-RC assembly. Loss-of-function mutations in the chromatin-modifying genes SIR2, and to a lesser extent in SIR3 and SIR4, suppressed the cdc6-4 temperature-sensitive lethality. This suppression was independent of the well-known silencing roles for the SIR proteins at the HM loci, at telomeres, or at the rDNA locus. A deletion of SIR2 uniquely rescued both the DNA synthesis defect of the cdc6-4 mutant and its severe plasmid instability phenotype for many origins. A SIR2 deletion suppressed additional initiation mutants affecting pre-RC assembly but not mutants that act subsequently. These findings suggest that Sir2p negatively regulates the initiation of DNA replication through a novel mechanism and reveal another connection between proteins that initiate DNA synthesis and those that establish silent heterochromatin in budding yeast.[Keywords: DNA replication; SIR2; deacetylase] Supplemental material is available at http://www.genesdev.org.
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.Eukaryotic RNA polymerase II (RNAP II) is a multisubunit enzyme that is responsible for transcription of all proteinencoding 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 Ј and  subunits of bacterial RNA polymerase (RNAP), respectively (23, 49). The yeast counterpart of the bacterial ␣ 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 ␣ 2 Ј 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...
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