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)

Sin mutations in Saccharomyces cerevisiae alleviate transcriptional defects that result from the inactivation of the yeast SWVI/SNF complex. We have investigated the structural and functional consequences for the nucleosome of Sin mutations in histone H3. We directly test the hypothesis that mutations in histone H3 leading to a SWI/SNF-independent (Sin) phenotype in yeast lead to nucleosomal destabilization. In certain instances this is shown to be true; however, nucleosomal destabilization does not always occur. Topoisomerase I-mediated relaxation of minichromosomes assembled with either mutant histone H3 or wild-type H3 together with histones H2A, H2B, and H4 indicates that DNA is constrained into nucleosomal structures containing either mutant or wild-type proteins. However, nucleosomes containing particular mutant H3 molecules (R116-H and T118-I) are more accessible to digestion by micrococcal nuclease and do not constrain DNA in a precise rotational position, as revealed by digestion with DNase I. This result establishes that Sin mutations in histone H3 located close to the dyad axis can destabilize histone-DNA contacts at the periphery of the nucleosome core. Other nucleosomes containing a distinct mutant H3 molecule (E105-K) associated with a Sin phenotype show very little change in nucleosome structure and stability compared to wild-type nucleosomes. Both mutant and wild-type nucleosomes continue to restrict the binding of either TATA-binding protein/transcription factor IIA (TFIIA) or the RNA polymerase III transcription machinery. Thus, different Sin mutations in histone H3 alter the stability of histone-DNA interactions to various extents in the nucleosome while maintaining the fundamental architecture of the nucleosome and contributing to a common Sin phenotype.
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PMID:Sin mutations of histone H3: influence on nucleosome core structure and function. 937 28

RNA polymerase I (Pol I) transcription in the yeast Saccharomyces cerevisiae is greatly stimulated in vivo and in vitro by the multiprotein complex, upstream activation factor (UAF). UAF binds tightly to the upstream element of the rDNA promoter, such that once bound (in vitro), UAF does not readily exchange onto a competing template. Of the polypeptides previously identified in purified UAF, three are encoded by genes required for Pol I transcription in vivo: RRN5, RRN9, and RRN10. Two others, p30 and p18, have remained uncharacterized. We report here that the N-terminal amino acid sequence, its mobility in gel electrophoresis, and the immunoreactivity of p18 shows that it is histone H3. In addition, histone H4 was found in UAF, and myc-tagged histone H4 could be used to affinity-purify UAF. Histones H2A and H2B were not detectable in UAF. These results suggest that histones H3 and H4 probably account for the strong binding of UAF to DNA and may offer a means by which general nuclear regulatory signals could be transmitted to Pol I.
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PMID:Histones H3 and H4 are components of upstream activation factor required for the high-level transcription of yeast rDNA by RNA polymerase I. 939 Oct 47

Mounting evidence suggests that eukaryotic RNA polymerases preassociate with multiple transcription factors in the absence of DNA, forming RNA polymerase holoenzyme complexes. We have purified an apparent RNA polymerase I (Pol I) holoenzyme from Xenopus laevis cells by sequential chromatography on five columns: DEAE-Sepharose, Biorex 70, Sephacryl S300, Mono Q, and DNA-cellulose. Single fractions from every column programmed accurate promoter-dependent transcription. Upon gel filtration chromatography, the Pol I holoenzyme elutes at a position overlapping the peak of Blue Dextran, suggesting a molecular mass in the range of approximately 2 MDa. Consistent with its large mass, Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gels reveal approximately 55 proteins in fractions purified to near homogeneity. Western blotting shows that TATA-binding protein precisely copurifies with holoenzyme activity, whereas the abundant Pol I transactivator upstream binding factor does not. Also copurifying with the holoenzyme are casein kinase II and a histone acetyltransferase activity with a substrate preference for histone H3. These results extend to Pol I the suggestion that signal transduction and chromatin-modifying activities are associated with eukaryotic RNA polymerases.
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PMID:Histone acetyltransferase and protein kinase activities copurify with a putative Xenopus RNA polymerase I holoenzyme self-sufficient for promoter-dependent transcription. 985 2

Human transcription factor IIIC (hTFIIIC) is a multisubunit complex that directly recognizes promoter elements and recruits TFIIIB and RNA polymerase III. Here we describe the cDNA cloning and characterization of the 90-kDa subunit (hTFIIIC90) that is present within a DNA-binding subcomplex (TFIIIC2) of TFIIIC. hTFIIIC90 has no specific homology to any of the known yeast TFIIIC subunits. Immunodepletion and immunoprecipitation studies indicate that hTFIIIC90 is a bona fide subunit of TFIIIC2 and absolutely required for RNA polymerase III transcription. hTFIIIC90 shows interactions with the hTFIIIC220, hTFIIIC110, and hTFIIIC63 subunits of TFIIIC, the hTFIIIB90 subunit of TFIIIB, and the human RPC39 (hRPC39) and hRPC62 subunits of an initiation-specific subcomplex of RNA polymerase III. These interactions may facilitate both TFIIIB and RNA polymerase III recruitment to the preinitiation complex by TFIIIC. We show that hTFIIIC90 has an intrinsic histone acetyltransferase activity with a substrate specificity for histone H3.
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PMID:The TFIIIC90 subunit of TFIIIC interacts with multiple components of the RNA polymerase III machinery and contains a histone-specific acetyltransferase activity. 1052 58

The nature of the interaction of coactivator proteins with transcriptionally active promoters in chromatin is a fundamental question in transcriptional regulation by RNA polymerase II. In this study, we used a biochemical approach to examine the functional association of the coactivator p300 with chromatin templates. Using in vitro transcription template competition assays, we observed that p300 forms a stable, template-committed complex with chromatin during the transcription process. The template commitment is dependent on the time of incubation of p300 with the chromatin template and occurs independently of the presence of a transcriptional activator protein. In studies examining interactions between p300 and chromatin, we found that p300 binds directly to chromatin and that the binding requires the p300 bromodomain, a conserved 110-amino-acid sequence found in many chromatin-associated proteins. Furthermore, we observed that the isolated p300 bromodomain binds directly to histones, preferentially to histone H3. However, the isolated p300 bromodomain does not bind to nucleosomal histones under the same assay conditions, suggesting that free histones and nucleosomal histones are not equivalent as binding substrates. Collectively, our results suggest that the stable association of p300 with chromatin is mediated, at least in part, by the bromodomain and is critically important for p300 function. Furthermore, our results suggest a model for p300 function that involves distinct activator-dependent targeting and activator-independent chromatin binding activities.
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PMID:p300 forms a stable, template-committed complex with chromatin: role for the bromodomain. 1135 96

Initiation of transcription of protein-encoding genes by RNA polymerase II (Pol II) was thought to require transcription factor TFIID, a complex comprised of the TATA box-binding protein (TBP) and TBP-associated factors (TAF(II)s). In the presence of TBP-free TAF(II) complex (TFTC), initiation of Pol II transcription can occur in the absence of TFIID. TFTC containing the GCN5 acetyltransferase acetylates histone H3 in a nucleosomal context. We have identified a 130 kDa subunit of TFTC (SAP130) that shares homology with the large subunit of UV-damaged DNA-binding factor. TFTC preferentially binds UV-irradiated DNA, UV-damaged DNA inhibits TFTC-mediated Pol II transcription and TFTC is recruited in parallel with the nucleotide excision repair protein XP-A to UV-damaged DNA. TFTC preferentially acetylates histone H3 in nucleosomes assembled on UV-damaged DNA. In agreement with this, strong histone H3 acetylation occurs in intact cells after UV irradiation. These results suggest that the access of DNA repair machinery to lesions within chromatin may be facilitated by TFTC via covalent modification of chromatin. Thus, our experiments reveal a molecular link between DNA damage recognition and chromatin modification.
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PMID:UV-damaged DNA-binding protein in the TFTC complex links DNA damage recognition to nucleosome acetylation. 1140 95

Mitogen-activated protein (MAP) kinase phosphatase 1 (MKP-1) has been shown to play a critical role in mediating the feedback control of MAP kinase cascades in a variety of cellular processes, including proliferation and stress responsiveness. Although MKP-1 expression is induced by a broad array of extracellular stimuli, the mechanisms mediating its induction remain poorly understood. Here we show that MKP-1 mRNA was potently induced by arsenite and ultraviolet light and modestly increased by heat shock and hydrogen peroxide. Interestingly, arsenite also dramatically induces phosphorylation-acetylation of histone H3 at a global level which precedes the induction of MKP-1 mRNA. The transcriptional induction of MKP-1, histone H3 modification, and elevation in MKP-1 mRNA in response to arsenite are all partially prevented by the p38 MAP kinase inhibitor SB203580, suggesting that the p38 pathway is involved in these processes. Finally, analysis of the DNA brought down by chromatin immunoprecipitation (ChIP) reveals that arsenite induces phosphorylation-acetylation of histone H3 associated with the MKP-1 gene and enhances binding of RNA polymerase II to MKP-1 chromatin. ChIP assays following exposure to other stress agents reveal various degrees of histone H3 modification at the MKP-1 chromatin. The differential contribution of p38 and ERK MAP kinases in mediating MKP-1 induction by different stress agents further illustrates the complexity and versatility of stress-induced MKP-1 expression. Our results strongly suggest that chromatin remodeling after stress contributes to the transcriptional induction of MKP-1.
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PMID:Transcriptional induction of MKP-1 in response to stress is associated with histone H3 phosphorylation-acetylation. 1168 10

The human ISWI-containing factor RSF (remodeling and spacing factor) was found to mediate nucleosome deposition and, in the presence of ATP, generate regularly spaced nucleosome arrays. Using this system, recombinant chromatin was reconstituted with bacterially produced histones. Acetylation of the histone tails was found to play an important role in establishing regularly spaced nucleosome arrays. Recombinant chromatin lacking histone acetylation was impaired in directing transcription. Histone-tail modifications were found to regulate transcription from the recombinant chromatin. Acetylation of the histone tails by p300 was found to increase transcription. Methylation of the histone H3 tail by Suv39H1 was found to repress transcription in an HP1-dependent manner. The effects of histone-tail modifications were observed in nuclear extracts. A highly reconstituted RNA polymerase II transcription system was refractory to the effect imposed by acetylation and methylation.
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PMID:Reconstitution of recombinant chromatin establishes a requirement for histone-tail modifications during chromatin assembly and transcription. 1169 35

Human Elongator complex was purified to virtual homogeneity from HeLa cell extracts. The purified factor can exist in two forms: a six-subunit complex, holo-Elongator, which has histone acetyltransferase activity directed against histone H3 and H4, and a three-subunit core form, which does not have histone acetyltransferase activity despite containing the catalytic Elp3 subunit. Elongator is a component of early elongation complexes formed in HeLa nuclear extracts and can interact directly with RNA polymerase II in solution. Several human homologues of the yeast Elongator subunits were identified as subunits of the human Elongator complex, including StIP1 (STAT-interacting protein 1) and IKAP (IKK complex-associated protein). Mutations in IKAP can result in the severe human disorder familial dysautonomia, raising the possibility that this disease might be due to compromised Elongator function and therefore could be a transcription disorder.
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PMID:Purification and characterization of the human elongator complex. 1171 25

Activation of gene transcription involves chromatin remodeling by coactivator proteins that are recruited by DNA-bound transcription factors. Local modification of chromatin structure at specific gene promoters by ATP-dependent processes and by posttranslational modifications of histone N-terminal tails provides access to RNA polymerase II and its accompanying transcription initiation complex. While the roles of lysine acetylation, serine phosphorylation, and lysine methylation of histones in chromatin remodeling are beginning to emerge, low levels of arginine methylation of histones have only recently been documented, and its physiological role is unknown. The coactivator CARM1 methylates histone H3 at Arg17 and Arg26 in vitro and cooperates synergistically with p160-type coactivators (e.g., GRIP1, SRC-1, ACTR) and coactivators with histone acetyltransferase activity (e.g., p300, CBP) to enhance gene activation by steroid and nuclear hormone receptors (NR) in transient transfection assays. In the current study, CARM1 cooperated with GRIP1 to enhance steroid hormone-dependent activation of stably integrated mouse mammary tumor virus (MMTV) promoters, and this coactivator function required the methyltransferase activity of CARM1. Chromatin immunoprecipitation assays and immunofluorescence studies indicated that CARM1 and the CARM1-methylated form of histone H3 specifically associated with a large tandem array of MMTV promoters in a hormone-dependent manner. Thus, arginine-specific histone methylation by CARM1 is an important part of the transcriptional activation process.
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PMID:Hormone-dependent, CARM1-directed, arginine-specific methylation of histone H3 on a steroid-regulated promoter. 1174 26


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