Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

mRNA capping is a cotranscriptional event mediated by the association of capping enzyme with the phosphorylated carboxy-terminal domain (CTD) of RNA polymerase II. In the yeast Saccharomyces cerevisiae, capping enzyme is composed of two subunits, the mRNA 5'-triphosphatase (Cet1) and the mRNA guanylyltransferase (Ceg1). Here we map interactions between Ceg1, Cet1, and the CTD. Although the guanylyltransferase subunit can bind alone to the CTD, it cannot be guanylylated unless the triphosphatase subunit is also present. Therefore, the yeast mRNA guanylyltransferase is regulated by allosteric interactions with both the triphosphatase and CTD.
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PMID:Allosteric interactions between capping enzyme subunits and the RNA polymerase II carboxy-terminal domain. 983 1

The activities of several mRNA processing factors are coupled to transcription through binding to RNA polymerase II (Pol II). The largest subunit of Pol II contains a repetitive carboxy-terminal domain (CTD) that becomes highly phosphorylated during transcription. mRNA-capping enzyme binds only to phosphorylated CTD, whereas other processing factors may bind to both phosphorylated and unphosphorylated forms. Capping occurs soon after transcription initiation and before other processing events, raising the question of whether capping components remain associated with the transcription complex after they have modified the 5' end of the mRNA. Chromatin immunoprecipitation in Saccharomyces cerevisiae shows that capping enzyme cross-links to promoters but not coding regions. In contrast, the mRNA cap methyltransferase and the Hrp1/CFIB polyadenylation factor cross-link to both promoter and coding regions. Remarkably, the phosphorylation pattern of the CTD changes during transcription. Ser 5 phosphorylation is detected primarily at promoter regions dependent on TFIIH. In contrast, Ser 2 phosphorylation is seen only in coding regions. These results suggest a dynamic association of mRNA processing factors with differently modified forms of the polymerase throughout the transcription cycle.
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PMID:Different phosphorylated forms of RNA polymerase II and associated mRNA processing factors during transcription. 1101 13

The Saccharomyces cerevisiae mRNA capping enzyme consists of two subunits: an RNA 5'-triphosphatase (Cet1) and an mRNA guanylyltransferase (Ceg1). In yeast, the capping enzyme is recruited to the RNA polymerase II (Pol II) transcription complex via an interaction between Ceg1 and the phosphorylated carboxy-terminal domain of the Pol II largest subunit. Previous in vitro experiments showed that the Cet1 carboxy-terminal region (amino acids 265 to 549) carries RNA triphosphatase activity, while the region containing amino acids 205 to 265 of Cet1 has two functions: it mediates dimerization with Ceg1, but it also allosterically activates Ceg1 guanylyltransferase activity in the context of Pol II binding. Here we characterize several Cet1 mutants in vivo. Mutations or deletions of Cet1 that disrupt interaction with Ceg1 are lethal, showing that this interaction is essential for proper capping enzyme function in vivo. Remarkably, the interaction region of Ceg1 becomes completely dispensable when Ceg1 is substituted by the mouse guanylyltransferase, which does not require allosteric activation by Cet1. Although no interaction between Cet1 and mouse guanylyltransferase is detectable, both proteins are present at yeast promoters in vivo. These results strongly suggest that the primary physiological role of the Ceg1-Cet1 interaction is to allosterically activate Ceg1, rather than to recruit Cet1 to the Pol II complex.
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PMID:The essential interaction between yeast mRNA capping enzyme subunits is not required for triphosphatase function in vivo. 1109 81

Using a highly pure transcription system derived from Saccharomyces cerevisiae, we have purified an activity in yeast whole-cell extracts that represses RNA polymerase II transcription. Mechanistic studies suggest that this repressor specifically targets transcriptional reinitiation. The two polypeptides that constitute the repressor have been identified as Ceg1p and Cet1p, the two subunits of the yeast pre-mRNA capping enzyme. A purified recombinant capping enzyme is able to reconstitute repressor activity. Cet1p is necessary for and capable of this repression. Transcriptional run-on experiments indicate that the capping enzyme also serves as a repressor in vivo. Efficient pre-mRNA capping relies on interactions between the capping enzyme and transcription apparatus. Repression by the capping enzyme suggests a bidirectional flow of information between capping and transcription.
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PMID:The yeast capping enzyme represses RNA polymerase II transcription. 1241 31

The Saccharomyces cerevisiae mRNA capping enzyme consists of two subunits: an RNA 5'-triphosphatase (RTPase) and GTP::mRNA guanylyltransferase (GTase). The GTase subunit (Ceg1) binds to the phosphorylated carboxyl-terminal domain of the largest subunit (CTD-P) of RNA polymerase II (pol II), coupling capping with transcription. Ceg1 bound to the CTD-P is inactive unless allosterically activated by interaction with the RTPase subunit (Cet1). For purposes of comparison, we characterize here the related GTases and RTPases from the yeasts Schizosaccharomyces pombe and Candida albicans. Surprisingly, the S. pombe capping enzyme subunits do not interact with each other. Both can independently interact with CTD-P of pol II, and the GTase is not repressed by CTD-P binding. The S. pombe RTPase gene (pct1+) is essential for viability. Pct1 can replace the S. cerevisiae RTPase when GTase activity is supplied by the S. pombe or mouse enzymes but not by the S. cerevisiae GTase. The C. albicans capping enzyme subunits do interact with each other. However, this interaction is not essential in vivo. Our results reveal an unexpected diversity among the fungal capping machineries.
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PMID:Divergent subunit interactions among fungal mRNA 5'-capping machineries. 1245 93

During transcription elongation, eukaryotic RNA polymerase II (Pol II) must contend with the barrier presented by nucleosomes. The conserved Spt4-Spt5 complex has been proposed to regulate elongation through nucleosomes by Pol II. To help define the mechanism of Spt5 function, we have characterized proteins that coimmunopurify with Spt5. Among these are the general elongation factors TFIIF and TFIIS as well as Spt6 and FACT, factors thought to regulate elongation through nucleosomes. Spt5 also coimmunopurified with the mRNA capping enzyme and cap methyltransferase, and spt4 and spt5 mutations displayed genetic interactions with mutations in capping enzyme genes. Additionally, we found that spt4 and spt5 mutations lead to accumulation of unspliced pre-mRNA. Spt5 also copurified with several previously unstudied proteins; we demonstrate that one of these is encoded by a new member of the SPT gene family. Finally, by immunoprecipitating these factors we found evidence that Spt5 participates in at least three Pol II complexes. These observations provide new evidence of roles for Spt4-Spt5 in pre-mRNA processing and transcription elongation.
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PMID:Dual roles for Spt5 in pre-mRNA processing and transcription elongation revealed by identification of Spt5-associated proteins. 1255 96

The Tup1-Ssn6 complex is an important corepressor in Saccharomyces cerevisiae that inhibits transcription through interactions with the basal transcription machinery and by remodeling chromatin. In a two-hybrid screen for factors that interact with the Schizosaccharomyces pombe Tup1 ortholog, Tup11, we isolated the pct1+ cDNA. The pct1+ gene encodes an mRNA 5'-triphosphatase, which catalyzes the first step of mRNA capping reactions. Pct1 did not interact with the S. pombe Ssn6 ortholog. In vitro glutathione S-transferase pull-down experiments revealed that Pct1 binds to the WD repeat regions of Tup11 and the functionally redundant Tup12 protein. Similarly, the S. cerevisiae Tup1 protein associates with the mRNA 5'-triphosphatase encoded by the CET1 gene. The highly conserved C-terminal domain of Cet1 interacts with Tup1 in vitro, and Tup1-Ssn6 complexes co-purify with the Cet1 protein, indicating that in vivo interactions also occur between these proteins. Over-expression of CET1 compromised repression of an MFA2-lacZ reporter gene that is subject to Tup1-Ssn6 repression. These genetic and biochemical interactions between Tup1-Ssn6 and Cet1 indicate that the capping enzyme associated with RNA polymerase II is a target of the corepressor complex.
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PMID:Physical and functional interaction of the yeast corepressor Tup1 with mRNA 5'-triphosphatase. 1263 15

Twenty-three chlorovirus genes expressed in host cells as early as 5-10 min postinfection (p.i.), or immediate early, were isolated and characterized. Some showed significant homology with those for transcriptional factors and mRNA-processing proteins including TFIIB, helicases, mRNA capping enzyme, nucleolin, and bean transcription factor. Others code for (i) factors influencing translation such as aminoacyl tRNA synthetases and ribosomal protein, and (ii) unknown proteins. Enzymes involved in polysaccharide synthesis were also found. All transcripts of these genes had a poly(A) tail, which decreased in size after 20 min p.i., possibly caused by the shortening by an exonuclease. Often, due to readthrough either from an upstream ORF or into a downstream ORF, a few extra transcripts for each gene appeared after 40 min p.i., suggesting a change in promoter selection and termination accuracy at this point. A typical TATA-box and a common element 5'-ATGACAA were in the promoter region of almost all of the immediate early genes, which may be recognized by host RNA polymerase and transcription factors.
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PMID:Immediate early genes expressed in chlorovirus infections. 1497 49

The structure of the Bluetongue virus (BTV) core and its outer layer VP7 has been solved by X-ray crystallography, but the assembly intermediates that lead to the inner scaffolding VP3 layer have not been defined. In this report, we addressed two key questions: (a) the role of VP3 amino terminus in core assembly and its interaction with the transcription complex (TC) components; and (b) the assembly intermediates involved in the construction of the VP3 shell. To do this, deletion mutants in the amino terminal and decamer-decamer interacting region of VP3 (DeltaDD) were generated, expressed in insect cells using baculovirus expression systems, and their ability to assemble into core-like particles (CLPs) and to incorporate the components of TC were investigated. Deletion of the N-terminal 5 (Delta5N) or 10 (Delta10N) amino acids did not affect the ability to assemble into CLPs in the presence of VP7 although the cores assembled using the 10 residue mutant (Delta10N) deletion were very unstable. Removal of five residues also did not effect incorporation of the internal VP1 RNA polymerase and VP4 mRNA capping enzyme proteins of the TC. Removal of the VP3-VP3 interacting domain (DeltaDD) led to failure to assemble into CLPs yet retained interaction with VP1 and VP4. In solution, purified DeltaDD mutant protein readily multimerized into dimers, pentamers, and decamers, suggesting that these oligomers are the authentic assembly intermediates of the subcore. However, unlike wild-type VP3 protein, the dimerization domain-deleted assembly intermediates were found to have lost RNA binding ability. Our study emphasizes the requirement of the N-terminus of VP3 for binding and encapsidation of the TC components, and defines the role of the dimerization domain in subcore assembly and RNA binding.
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PMID:Mapping the assembly pathway of Bluetongue virus scaffolding protein VP3. 1520 24

Mdm30p, a nucleus-encoded F-box protein, which binds to the substrate for ubiquitin-mediated proteolysis, is involved in maintenance of fusion-competent mitochondria for various cellular functions. Recently, Mdm30p has been implicated in regulation of gene expression. However, its mode of action in gene regulation is not clearly known in vivo. With this view, we have systematically analyzed here the role of Mdm30p in regulation of transcriptional initiation, elongation, mRNA processing, and export in Saccharomyces cerevisiae, using a formaldehyde-based in vivo cross-linking and chromatin immunoprecipitation assay in conjunction with RT-PCR and fluorescence in situ hybridization. We show that Mdm30p is dispensable for formation of the preinitiation complex assembly, association of elongating RNA polymerase II, and recruitment of mRNA capping enzyme, cap-binding complex, and 3' end formation machinery at the transcriptionally active genes such as ADH1, PHO84, and RPS5. Intriguingly, we find that Mdm30p facilitates the recruitment of the transcription-export complex at these genes. Consistently, the export of mRNAs of these genes is significantly impaired in the absence of Mdm30p as revealed by fluorescence in situ hybridization and RT-PCR analysis of cytoplasmic mRNA. However, such an impaired mRNA export is not dependent on mitochondrial fusion, as the deletion of FZO1, an essential gene for mitochondrial fusion, does not alter the export of ADH1, PHO84, and RPS5 mRNAs. Together, our data demonstrate that Mdm30p selectively controls mRNA export independently of mitochondrial fusion, revealing a novel function of an F-box protein in mRNA export.
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PMID:Stimulation of mRNA export by an F-box protein, Mdm30p, in vivo. 1937 28


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