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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)

Recent studies from a number of laboratories have revealed a surprising number of connections between RNA polymerase II transcription and the ubiquitin/proteasome pathway. We now find yet another intersection of these pathways by showing that the 26S proteasome associates with regions of the GAL1, GAL10, and HSP82 genes, including the 3' ends, in a transcription-dependent fashion. The appearance of the proteasome on these inducible genes correlates with both the accumulation of transcripts and the buildup of RNA polymerase II complexes in the same region. Furthermore, the 26S proteasome and RNA polymerase II coimmunoprecipitate, and inhibition of 26S proteolytic activity leads to increased read through of a transcription termination site. We suggest that the proteasome is generally recruited to the DNA at sites of stalled RNA polymerase and may act to resolve these complexes.
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PMID:Physical and functional association of RNA polymerase II and the proteasome. 1506 96

One of the temperature-sensitive alleles of CEG1, a guanylyltransferase subunit of the Saccharomyces cerevisiae capping enzyme, showed 6-azauracil (6AU) sensitivity at the permissive growth temperature, which is a phenotype that is correlated with a transcription elongation defect. This temperature-sensitive allele, ceg1-63, has an impaired ability to induce PUR5 in response to 6AU treatment and diminished enzyme-GMP formation activity. However, this cellular and molecular defect is not primarily due to the preferential degradation of the transcript attributed to a lack of cap structure. Our data suggest that the guanylyltransferase subunit of the capping enzyme plays a role in transcription elongation as well as cap formation. First, in addition to the 6AU sensitivity, ceg1-63 is synthetically lethal with elongation-defective mutations in RNA polymerase II. Secondly, it produces a prolonged steady-state level of GAL1 mRNA after glucose shutoff. Third, it decreases the transcription read through a tandem array of promoter-proximal pause sites in an orientation-dependent manner. Taken together, we present direct evidence that suggests a role of capping enzyme in an early transcription. Capping enzyme ensures the early transcription checkpoint by capping of the nascent transcript in time and allowing it to extend further.
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PMID:mRNA capping enzyme activity is coupled to an early transcription elongation. 1522 22

Mechanistic analysis of transcriptional initiation and termination by RNA polymerase II (PolII) indicates that some factors are common to both processes. Here we show that two long genes of Saccharomyces cerevisiae, FMP27 and SEN1, exist in a looped conformation, effectively bringing together their promoter and terminator regions. We also show that PolII is located at both ends of FMP27 when this gene is transcribed from a GAL1 promoter under induced and noninduced conditions. Under these conditions, the C-terminal domain of the large subunit of PolII is phosphorylated at Ser5. Notably, inactivation of Kin28p causes a loss of both Ser5 phosphorylation and the loop conformation. These data suggest that gene loops are involved in the early stages of transcriptional activation. They also predict a previously unknown structural dimension to gene regulation, in which both ends of the transcription unit are defined before and during the transcription cycle.
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PMID:Gene loops juxtapose promoters and terminators in yeast. 1531 41

The chromatin remodeling activity of the Swi/Snf complex is essential for the expression of several yeast genes. Previous studies have suggested that recruitment of Swi/Snf requires the action of transcriptional activators. However, reports in metazoans and in yeast have provided evidence of interactions between Swi/Snf and the RNA polymerase II holoenzyme/Mediator complex. Here we show that recruitment of Swi/Snf to the galactose-inducible gene GAL1 cannot be fully achieved without the integrity of the Mediator complex, TAF IIs, and RNA polymerase II. Moreover, artificial recruitment of Mediator is sufficient to tether both Swi/Snf and SAGA to the GAL1 UAS G. We further demonstrate that Swi/Snf recruitment at GAL1 does not require acetylation of chromatin by Gcn5 nor the presence of SAGA. Based on these results, we conclude that interactions between the Gal4 activator and Swi/Snf are not sufficient to recruit the latter to the GAL1 UAS G, since interactions with the Mediator, TAF IIs, and RNA polymerase II are also important.
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PMID:Targeting of Swi/Snf to the yeast GAL1 UAS G requires the Mediator, TAF IIs, and RNA polymerase II. 1538 57

Rad6-mediated ubiquitylation of histone H2B at lysine 123 has been linked to transcriptional activation and the regulation of lysine methylation on histone H3. However, how Rad6 and H2B ubiquitylation contribute to the transcription and histone methylation processes is poorly understood. Here, we show that the Paf1 transcription elongation complex and the E3 ligase for Rad6, Bre1, mediate an association of Rad6 with the hyperphosphorylated (elongating) form of RNA polymerase II (Pol II). This association appears to be necessary for the transcriptional activities of Rad6, as deletion of various Paf1 complex members or Bre1 abolishes H2B ubiquitylation (ubH2B) and reduces the recruitment of Rad6 to the promoters and transcribed regions of active genes. Using the inducible GAL1 gene as a model, we find that the recruitment of Rad6 upon activation occurs rapidly and transiently across the gene and coincides precisely with the appearance of Pol II. Significantly, during GAL1 activation in an rtf1 deletion mutant, Rad6 accumulates at the promoter but is absent from the transcribed region. This fact suggests that Rad6 is recruited to promoters independently of the Paf1 complex but then requires this complex for entrance into the coding region of genes in a Pol II-associated manner. In support of a role for Rad6-dependent H2B ubiquitylation in transcription elongation, we find that ubH2B levels are dramatically reduced in strains bearing mutations of the Pol II C-terminal domain (CTD) and abolished by inactivation of Kin28, the serine 5 CTD kinase that promotes the transition from initiation to elongation. Furthermore, synthetic genetic array analysis reveals that the Rad6 complex interacts genetically with a number of known or suspected transcription elongation factors. Finally, we show that Saccharomyces cerevisiae mutants bearing defects in the pathway to H2B ubiquitylation display transcription elongation defects as assayed by 6-azauracil sensitivity. Collectively, our results indicate a role for Rad6 and H2B ubiquitylation during the elongation cycle of transcription and suggest a mechanism by which H3 methylation may be regulated.
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PMID:Histone H2B ubiquitylation is associated with elongating RNA polymerase II. 1563 65

TFIIS is a transcription elongation factor that has been extensively studied biochemically. Although the in vitro mechanisms by which TFIIS stimulates RNA transcript cleavage and polymerase read-through have been well characterized, its in vivo roles remain unclear. To better understand TFIIS function in vivo, we have examined its role during Gal4-mediated activation of the Saccharomyces cerevisiae GAL1 gene. Surprisingly, TFIIS is strongly associated with the GAL1 upstream activating sequence. In addition, TFIIS recruitment to Gal4-binding sites is dependent on Gal4, SAGA, and Mediator but not on RNA polymerase II (Pol II). The association of TFIIS is also necessary for the optimal recruitment of TATA-binding protein and Pol II to the GAL1 promoter. These results provide strong evidence that TFIIS plays an important role in the initiation of transcription at GAL1 in addition to its well-characterized roles in transcription elongation.
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PMID:Evidence that the elongation factor TFIIS plays a role in transcription initiation at GAL1 in Saccharomyces cerevisiae. 1576 71

Previous biochemical studies have demonstrated that Lys-123 ubiquitination of histone H2B is globally required for up-regulation of mono-, di, and trimethylation of Lys-4 of histone H3. However, recent studies have implicated H2B-Lys-123 ubiquitination in the regulation of di- and trimethylation, but not monomethylation, of H3-Lys-4 in vivo. Using a formaldehyde-based cross-linking and chromatin immunoprecipitation assay, we show that H3-Lys-4 trimethylation, but not dimethylation, is up-regulated by H2B-Lys-123 ubiquitination in vivo at the coding sequences of a set of transcriptionally active genes such as ADH1, PHO84, and PYK1. Both the ubiquitination of H2B-Lys-123 and the methylation of H3-Lys-4 are dispensable for recruitment of RNA polymerase II to the coding sequences of these genes, and hence, their transcription is not altered in the absence of these covalent modifications. However, recruitment of RNA polymerase II to the coding sequence of a galactose-inducible gene, GAL1, is significantly reduced in the absence of H2B-Lys-123 ubiquitination but not H3-Lys-4 methylation. Consistently, transcription of GAL1 is altered in the H2B-K123R point mutant strain. Finally, we show that H3-Lys-4 methylation does not regulate H3-Lys-9/14 acetylation. Collectively, our data reveal a "trans-tail" regulation of H3-Lys-4 tri- but not dimethylation by H2B-Lys-123 ubiquitination, and these modifications are dispensable for transcription of a certain set of genes in vivo.
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PMID:Functional analysis of H2B-Lys-123 ubiquitination in regulation of H3-Lys-4 methylation and recruitment of RNA polymerase II at the coding sequences of several active genes in vivo. 1667 45

The FACT complex stimulates transcription elongation on nucleosomal templates. In vivo experiments also involve FACT in the reassembly of nucleosomes traversed by RNA polymerase II. Since several features of chromatin organization vary throughout the genome, we wondered whether FACT is equally required for all genes. We show in this study that the in vivo depletion of Spt16, one of the subunits of Saccharomyces cerevisiae FACT, strongly affects transcription of three genes, GAL1, PHO5, and Kluyveromyces lactis LAC4, which exhibit positioned nucleosomes at their transcribed regions. In contrast, showing a random nucleosome structure, YAT1 and Escherichia coli lacZ are only mildly influenced by Spt16 depletion. We also show that the effect of Spt16 depletion on GAL1 expression is suppressed by a histone mutation and that the insertion of a GAL1 fragment, which allows the positioning of two nucleosomes, at the 5' end of YAT1 makes the resulting transcription unit sensitive to Spt16 depletion. These results indicate that FACT requirement for transcription depends on the chromatin organization of the 5' end of the transcribed region.
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PMID:A gene-specific requirement for FACT during transcription is related to the chromatin organization of the transcribed region. 1700 Jul 68

Rad26, the yeast homologue of human Cockayne syndrome group B protein, and Rpb9, a nonessential subunit of RNA polymerase II, have been shown to mediate two subpathways of transcription-coupled DNA repair in yeast. Here we show that Rad26- and Rpb9-mediated repair in the yeast GAL1 gene is differently modulated by different promoter elements. The initiation site and efficiency of Rad26-mediated repair in the transcribed strand are determined by the upstream activating sequence (UAS) but not by the TATA or local sequences. The role of UAS in determining the Rad26-mediated repair is not through loading of RNA polymerase II or the transcriptional regulatory complex SAGA. However, both the UAS and the TATA sequences are essential for confining Rad26-mediated repair to the transcribed strand. Mutation of the TATA sequence, which greatly reduces transcription, or deletion of the TATA or mutation of the UAS, which completely abolishes transcription, causes Rad26-mediated repair to occur in both strands. Rpb9-mediated repair only occurs in the transcribed strand and is efficient only in the presence of both TATA and UAS sequences. Also, the efficiency of Rpb9-mediated repair is dependent on the SAGA complex. Our results suggest that Rad26-mediated repair can be either transcription-coupled, provided that a substantial level of transcription is present, or transcription-independent, if the transcription is too low or absent. In contrast, Rpb9-mediated repair is strictly transcription-coupled and is efficient only when the transcription level is high.
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PMID:Modulation of Rad26- and Rpb9-mediated DNA repair by different promoter elements. 1702 24

Eukaryotic gene regulation is closely correlated with histone covalent modifications. Recently, histone H2B lysine-123 (H2B-K123) ubiquitination has been implicated in regulation of transcription as well as histone H3 lysine-4 (H3-K4) methylation which is further associated with active transcription. However, whether H2B-K123 ubiquitination controls transcription through regulation of H3-K4 methylation remains unknown under physiological conditions. Here, we show that H2B-K123 ubiquitination enhances the rate of elongating RNA polymerase II (RNAPII) recruitment to the coding sequence of an inducible yeast gene, GAL1. Consistently, GAL1 transcription is significantly impaired in absence of H2B-K123 ubiquitination. On the other hand, H3-K4 methylation does not alter the rate of elongating RNAPII recruitment at GAL1. Further, these covalent modifications do not regulate pre-initiation complex formation at GAL1. Collectively, our data demonstrate the function of H2B-K123 ubiquitination in regulation of transcriptional elongation independently of H3-K4 methylation in vivo, providing a new insight on epigenetic regulation of gene expression.
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PMID:H2B-K123 ubiquitination stimulates RNAPII elongation independent of H3-K4 methylation. 1754 90


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