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)

In eukaryotic cells, genomic DNA is assembled with chromosomal proteins, mainly histones, in a highly compact structure termed chromatin. In this form, DNA is not readily accessible to the cellular machineries, which require DNA as a template. Dynamic changes in chromatin organization play a critical role in regulation of DNA-dependent processes such as transcription, DNA replication, recombination and repair. Chromatin structure is altered in transcriptionally active loci: the basic chromatin unit, the nucleosome, appears to be depleted for one histone H2A/H2B dimer. Previously, reconstitution of RNA polymerase II (PolII)-driven transcription on chromatin templates in a highly purified in vitro system led to identification of FACT (for facilitates chromatin transcription), which was required for productive transcript elongation through nucleosomes. FACT was proposed to promote PolII transcription through nucleosomes by removing either one or both H2A/H2B dimers. Here we present an overview of the earlier studies, which resulted in the initial identification and characterization of FACT, as well as the recent findings that refine the model for the mechanism of FACT function in transcription.
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PMID:Transcription through chromatin: understanding a complex FACT. 1502 50

Transcription through the nucleosome by Saccharomyces cerevisiae RNA polymerase II (Pol II) is characterized by an almost absolute block to transcription at physiological ionic strength and displacement of one H2A/H2B dimer to form a hexasome [Mol. Cell 9 (2002) 541]. In previous studies of Pol II transcription through chromatin, templates containing nucleosomes in multiple positions were used. These templates do not allow detailed analysis of the mechanism of transcription through chromatin. Here, we describe the development of a new template that is only long enough to accommodate a single nucleosome position along the DNA so that all of the templates are identical and allow for more in-depth analysis. After ligation of the nucleosome to promoter DNA or assembled elongation complexes, the mechanism of transcription through this uniquely positioned nucleosome by various RNA polymerases can be analyzed.
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PMID:Construction, analysis, and transcription of model nucleosomal templates. 1503 83

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

Both indirect (transcription-induced stress) and direct effects of polymerase elongation on histone-DNA interactions were studied on closed circular DNA that was either moderately or positively coiled. The templates were reconstituted with (3)H-labeled H2A, H2B, H3, and H4 to form nucleosomes, and transcription was done with T7 RNA polymerase in the presence of a negatively coiled competitor DNA (reconstituted with unlabeled H3 and H4). The first of the two labeled H2A-H2B dimers readily displaced from the highly positively coiled template to the competitor even in the absence of transcription, while the indirect effect of transcription-induced stress was required for the moderately coiled template. The second labeled H2A-H2B dimer required transcription-induced stress for both moderately and highly positively coiled DNA. The displacement of the labeled H3-H4 tetramer also occurred, provided it was associated with an H2A-H2B dimer and a moderately positively coiled DNA. This displacement occurred independent of transcription-induced stress and is likely due to the direct effect of polymerase disruption of histone-DNA interactions. The inclusion of the histone chaperone, NAP1, greatly enhanced the release of both of the two H2A-H2B dimers. These observations are consistent with in vivo observations which indicate that during transcription H2A and H2B are significantly more mobile than H3 and H4 and indicate that transcription-induced positive stress is a likely cause for this selective movement.
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PMID:Histone release during transcription: displacement of the two H2A-H2B dimers in the nucleosome is dependent on different levels of transcription-induced positive stress. 1580 29

The post-translational modification of histones and the incorporation of core histone variants play key roles in governing gene expression. Many eukaryotic genes regulate their expression by limiting the escape of RNA polymerase from promoter-proximal pause sites. Here we report that elongating RNA polymerase II complexes encounter distinct chromatin landscapes that are marked by methylation of lysine residues Lys(4), Lys(79), and Lys(36) of histone H3. However, neither histone methylation nor acetylation directly regulates the release of elongation complexes stalled at promoter-proximal pause sites of the c-myc gene. In contrast, transcriptional activation is associated with local displacement of the histone variant H2A.Z within the transcribed region and incorporation of the major histone variant H2A. This result indicates that transcribing RNA polymerase II remodels chromatin in part through coincident displacement of H2A.Z-H2B dimers and incorporation of H2A-H2B dimers. In combination, these results suggest a new model in which the incorporation of H2A.Z into nucleosomes down-regulates transcription; at the same time it may act as a cellular memory for transcriptionally poised gene domains.
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PMID:Transcription-induced chromatin remodeling at the c-myc gene involves the local exchange of histone H2A.Z. 1587 76

Histone-lysine methylation is linked to transcriptional regulation and the control of epigenetic inheritance. Lysine residues can be mono-, di-, or trimethylated, and it has been suggested that each methylation state of a given lysine may impart a unique biological function. In yeast, histone H3 lysine 4 (K4) is mono-, di-, and trimethylated by the Set1 histone methyltransferase. Previous studies show that Set1 associates with RNA polymerase II and demarcates transcriptionally active genes with K4 trimethylation. To determine whether K4 trimethylation might be selectively regulated, we screened a library of yeast deletion mutants associated with transcriptional regulation and chromatin function. We identified BUR2, a cyclin for the Bur1/2 (BUR) cyclin-dependent protein kinase, as a specific regulator of K4 trimethylation. Surprisingly, BUR also regulated H2B monoubiquitylation, whereas other K4 methylation states and H3 lysine 79 (K79) methylation were unaffected. Synthetic genetic array (SGA) and transcription microarray analyses of a BUR2 mutant revealed that BUR is functionally similar to the PAF, Rad6, and Set1 complexes. These data suggest that BUR acts upstream of these factors to control their function. In support, we show that recruitment of the PAF elongation complex to genes is significantly impaired in a BUR2 deletion. Our data reveal a novel function for the BUR kinase in transcriptional regulation through the selective control of histone modifications.
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PMID:BUR kinase selectively regulates H3 K4 trimethylation and H2B ubiquitylation through recruitment of the PAF elongation complex. 1604 Feb 46

A high level of the post-translational modification, acetylation, is found on the N-terminal regions of the core histones H2A, H2B, H3, and H4 and is primarily located in the nucleosomes of active genes. An in vitro transcription system was applied, which utilizes T7 RNA polymerase and template DNAs that are either moderately or highly positively coiled, to determine whether acetylation alters the dynamics of histone displacement from these templates during transcription. To measure displacement, an excess of a competitor (negatively coiled DNA reconstituted with unlabeled H3-H4) was included during the transcription process. Acetylated but not unacetylated (3)H-labeled H3-H4 was found to displace with high frequency from the moderately positively coiled template. This displacement of acetylated H3-H4 was not observed when the template was highly positively coiled. Acetylated (3)H-labeled H2A-H2B also preferentially displaced to the competitor, but in this instance, transcription-induced stress on the highly positively coiled template was required. The histone chaperone, NAP1, was found to facilitate the displacement of both H3-H4 and H2A-H2B. Surprisingly, when acetylated H2A-H2B and acetylated H3-H4 were reconstituted together in the same nucleosomes, the displacement of acetylated H2A-H2B was much reduced during transcription. We conclude that acetylation alters nucleosome stability by enhancing displacement of H3-H4, while decreasing the displacement of H2A-H2B. These results are discussed with regard to potential in vivo conditions in which these observations may be relevant.
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PMID:Histone release during transcription: acetylation stabilizes the interaction of the H2A-H2B dimer with the H3-H4 tetramer in nucleosomes that are on highly positively coiled DNA. 1633 96

Crystals of native histone octamers (H2A-H2B)-(H4-H3)-(H3'-H4')-(H2B'-H2A') from chick erythrocytes in 2 M KCl, 1.35 M potassium phosphate pH 6.9 diffract X-rays to 1.90 A resolution, yielding a structure with an R(work) value of 18.7% and an Rfree of 22.2%. The crystal space group is P6(5), the asymmetric unit of which contains one complete octamer. This high-resolution model of the histone-core octamer allows further insight into intermolecular interactions, including water molecules, that dock the histone dimers to the tetramer in the nucleosome-core particle and have relevance to nucleosome remodelling. The three key areas analysed are the H2A'-H3-H4 molecular cluster (also H2A-H3'-H4'), the H4-H2B' interaction (also H4'-H2B) and the H2A'-H4 beta-sheet interaction (also H2A-H4'). The latter of these three regions is important to nucleosome remodelling by RNA polymerase II, as it is shown to be a likely core-histone binding site, and its disruption creates an instability in the nucleosome-core particle. A majority of the water molecules in the high-resolution octamer have positions that correlate to similar positions in the high-resolution nucleosome-core particle structure, suggesting that the high-resolution octamer model can be used for comparative studies with the high-resolution nucleosome-core particle.
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PMID:High-resolution structure of the native histone octamer. 1651 Oct 91

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

Histones are rapidly evicted and deposited during transcription by RNA polymerase (Pol) II, but a factor that mediates histone eviction in vivo has not yet been identified. Here, we show that the histone chaperone Asf1 associates with promoters and coding regions of transcriptionally active genes. Asf1 mediates histone H3, but not H2B, eviction and deposition during Pol II elongation, suggesting that nucleosome assembly and disassembly occur in a stepwise fashion. Lastly, Asf1 inhibits internal initiation from cryptic promoters within coding regions. These results strongly suggest that Asf1 functions as an elongation factor to disassemble and reassemble histones during Pol II elongation.
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PMID:Asf1 mediates histone eviction and deposition during elongation by RNA polymerase II. 1667 13


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