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)

Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] has been known to bind to the pleckstrin homology domain and the phosphotyrosine-binding domain as well as actin-binding proteins, and to regulate their functions. We have tried to find new PtdIns(4,5)P2-binding proteins and to clarify the physiological effects of PtdIns(4,5)P2 on their function. We report here that histones H1 and H3 are PtdIns(4,5)P2-binding proteins which were identified using antibodies specific to PtdIns(4,5)P2, H1, and H3. This binding was further confirmed by extracting PtdIns(4,5)P2 from purified histone H1 and H3. Furthermore, the binding site of PtdIns(4,5)P2 in histone H1 was found in the carboxyl-terminal 103 amino acids. It was also shown that the amounts of PtdIns(4,5)P2 bound to H1 decrease when histone H1 is phosphorylated by protein kinase C but not by protein kinase A or cdc2 kinase, in vitro. The protein kinase C phosphorylation site is localized close to the PtdIns(4,5)P2-binding site, suggesting that phosphorylation of histone H1 by protein kinase C interferes stereostructurally with PtdIns(4,5)P2 binding. We further noticed that PtdIns(4,5)P2 binding to H1 counteracts the histone H1-mediated repression of basal transcription by RNA polymerase II in a Drosophila transcription system in vitro. Phosphatidylinositol 4-phosphate and phosphatidylinositol 3,4,5-trisphosphate affect this transcription activity more weakly than PtdIns(4,5)P2, but PtdIns and other acidic lipids have no effect on this activity. These data indicate that PtdIns(4,5)P2 bound to nuclear protein histone H1 may contribute to the regulation of transcription in eukaryotic cells.
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PMID:Phosphatidylinositol 4,5-bisphosphate reverses the inhibition of RNA transcription caused by histone H1. 949 95

The activation of cyclin-dependent kinases (CDKs) requires phosphorylation of a threonine residue within the T-loop catalyzed by CDK-activating kinases (CAKs). Thus far no functional CAK homologue has been reported in plants. We screened an Arabidopsis cDNA expression library for complementation of a budding yeast CAK mutant. A cDNA, cak1At, was isolated that suppressed the CAK mutation in budding yeast, and it also complemented a fission yeast CAK mutant. cak1At encodes a protein related to animal CAKs. The CAK similarity was restricted to the conserved kinase domains, leading to classification of Cak1At as a distinct CDK in the phylogenetic tree. Immunoprecipitates with the anti-Cak1At antibody phosphorylated human CDK2 at the threonine residue (T160) within the T-loop and activated its activity to phosphorylate histone H1. Whereas CAKs in animals and fission yeast are involved in regulation of the cell cycle and basal transcription by phosphorylating the carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II, Cak1At did not phosphorylate the CTD. An Arabidopsis CTD-kinase isolated separately from Cak1At was shown to interact with the yeast protein p13(suc1), but it had no CDK2-kinase activity. Therefore, the CTD of RNA polymerase II is probably phosphorylated by a Cdc2-related kinase distinct from Cak1At. cak1At is a single-copy gene in Arabidopsis and is highly expressed in proliferating cells of suspension cultures.
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PMID:A distinct cyclin-dependent kinase-activating kinase of Arabidopsis thaliana. 956 Feb 21

It has long been proposed that selective binding of histone H1 is, in part, responsible for the differential developmental regulation of the oocyte and somatic 5S rRNA genes in Xenopus laevis. In this study we show that histone H1 binds both oocyte and somatic genes equally after reconstitution into mononucleosomes or oligonucleosome arrays. Furthermore, we show that the binding of histone H1 selectively represses only oocyte gene transcription and that an RNA polymerase III transcription complex is able to initiate transcription of nucleosomal somatic templates regardless of whether histone H1 is present. These results support a model in which the differential regulation of the 5S rRNA genes is not simply due to the prevention of histone H1 binding by transcription complexes on the somatic genes, but rather to a difference in the histone H1 interaction with the somatic and oocyte genes.
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PMID:Histone H1 binding does not inhibit transcription of nucleosomal Xenopus laevis somatic 5S rRNA templates. 958 17

Several factors were evaluated to determine their role in facilitating the presence of transcription-induced stresses in a circular DNA. Transcription was done with T7 RNA polymerase in the presence of E. coli topoisomerase I and closed circular DNA. Positive stress was observed in hypotonic conditions or when one of the polyamines, spermidine or spermine, were present. Polycations such as polylysine, polyarginine, histone H1, histones H2A-H2B, and protamine were observed to induce minimal positive stress. It is known that polyamines influence DNA structure by causing both self-association and sequence-specific structural alterations (polyamine-induced localized bending). Experimental evidence indicates that the likely cause of the positive stress is the induced bending. In order to evaluate protein-mediated bending, transcription was done on nucleosomes. A minimum of three nucleosomes on a DNA of 6055 bp was sufficient to generate very high levels of positive stress. Histones H3-H4 in the absence of H2A-H2B were responsible for this effect. Since these histones by themselves are able to maintain negative coils on DNA, it is concluded that protein-mediated bending is yet another mechanism for placing rotational restriction on DNA. The bending of DNA by either polyamines or histones is an effective mechanism for promoting transcription-induced stresses at physiological ionic strength.
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PMID:In vitro studies on the maintenance of transcription-induced stress by histones and polyamines. 1061 64

Cyclin-dependent kinases (CDKs) that control cell cycle progression are regulated in many ways, including activating phosphorylation of a conserved threonine residue. This essential phosphorylation is carried out by the CDK-activating kinase (CAK). Here we examine the effects of replacing this threonine residue in human CDK2 by serine. We found that cyclin A bound equally well to wild-type CDK2 (CDK2(Thr-160)) or to the mutant CDK2 (CDK2(Ser-160)). In the absence of activating phosphorylation, CDK2(Ser-160)-cyclin A complexes were more active than wild-type CDK2(Thr-160)-cyclin A complexes. In contrast, following activating phosphorylation, CDK2(Ser-160)-cyclin A complexes were less active than phosphorylated CDK2(Thr-160)-cyclin A complexes, reflecting a much smaller effect of activating phosphorylation on CDK2(Ser-160). The kinetic parameters for phosphorylating histone H1 were similar for mutant and wild-type CDK2, ruling out a general defect in catalytic activity. Interestingly, the CDK2(Ser-160) mutant was selectively defective in phosphorylating a peptide derived from the C-terminal domain of RNA polymerase II. CDK2(Ser-160) was efficiently phosphorylated by CAKs, both human p40(MO15)(CDK7)-cyclin H and budding yeast Cak1p. In fact, the k(cat) values for phosphorylation of CDK2(Ser-160) were significantly higher than for phosphorylation of CDK2(Thr-160), indicating that CDK2(Ser-160) is actually phosphorylated more efficiently than wild-type CDK2. In contrast, dephosphorylation proceeded more slowly with CDK2(Ser-160) than with wild-type CDK2, either in HeLa cell extract or by purified PP2Cbeta. Combined with the more efficient phosphorylation of CDK2(Ser-160) by CAK, we suggest that one reason for the conservation of threonine as the site of activating phosphorylation may be to favor unphosphorylated CDKs following the degradation of cyclins.
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PMID:The effects of changing the site of activating phosphorylation in CDK2 from threonine to serine. 1093 29

The tandemly organised ribosomal DNA (rDNA) repeats are transcribed by a dedicated RNA polymerase in a specialised nuclear compartment, the nucleolus. There appears to be an intimate link between the maintenance of nucleolar structure and the presence of heterochromatic chromatin domains. This is particularly evident in many large neurons, where a single nucleolus is present, which is separated from the remainder of the nucleus by a characteristic shell of heterochromatin. Using a combined fluorescence in situ hybridisation and immunocytochemistry approach, we have analysed the molecular composition of this highly organised neuronal chromatin, to investigate its functional significance. We find that clusters of inactive, methylated rDNA repeats are present inside large neuronal nucleoli, which are often attached to the shell of heterochromatic DNA. Surprisingly, the methylated DNA-binding protein MeCP2, which is abundantly present in the centromeric and perinucleolar heterochromatin, does not associate significantly with the methylated rDNA repeats, whereas histone H1 does overlap partially with these clusters. Histone H1 also defines other, centromere-associated chromatin subdomains, together with the mammalian Polycomb group factor Eed. These data indicate that neuronal, perinucleolar heterochromatin consists of several classes of inactive DNA, that are linked to a fraction of the inactive rDNA repeats. These distinct chromatin domains may serve to regulate RNA transcription and processing efficiently and to protect rDNA repeats against unwanted silencing and/or homologous recombination events.
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PMID:Characterisation of transcriptionally active and inactive chromatin domains in neurons. 1108 40

p70ik3-1 (a 70-kDa protein) contains a cyclin box, and binds to p35cdk3 in vivo and in vitro [Matsuoka, M., Matsuura, Y., Semba, K. & Nishimoto, I. (2000) Biochem. Biophys. Res. Commun. 273, 442-447]. In spite of its structural similarity to cyclins, p70ik3-1 does not activate cyclin-dependent kinase 3 (cdk3)-mediated phosphorylation of pRb, histone H1, or the C-terminal domain of RNA polymerase II. Here, we report that Ser274 of p70ik3-1 is phosphorylated by cdk2 or cdk3 bound to cyclin A and to cyclin E in vitro. We also found that in COS7 cells in which cyclin E and cdk3 were ectopically overexpressed, the phosphorylation level of Ser274 in coexpressed p70ik3-1 is upregulated. We therefore conclude that p70ik3-1 is a substrate for cdk3-mediated phosphorylation.
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PMID:ik3-1/Cables is a substrate for cyclin-dependent kinase 3 (cdk 3). 1173 1

Chromatin is the physiological template for many nuclear processes in eukaryotes, including transcription by RNA polymerase II. In vivo, chromatin is assembled from genomic DNA, core histones, linker histones such as histone H1, and nonhistone chromatin-associated proteins. Histone H1 is thought to act as a general repressor of transcription by promoting the compaction of chromatin into higher-order structures. We have used a biochemical approach, including an in vitro chromatin assembly and transcription system, to examine the effects of histone H1 on estrogen receptor alpha (ER alpha)-mediated transcription with chromatin templates. We show that histone H1 acts as a potent repressor of ligand- and coactivator-regulated transcription by ER alpha. Histone H1 exerts its repressive effect without inhibiting the sequence-specific binding of ER alpha to chromatin or the overall extent of targeted acetylation of nucleosomal histones by the coactivator p300. Instead, histone H1 acts by blocking a specific step in the ER alpha-dependent transcription process, namely, transcription initiation, without affecting transcription reinitiation. Together, our data indicate that histone H1 acts selectively to reduce the overall level of productive transcription initiation by restricting promoter accessibility and preventing the ER alpha-dependent formation of a stable transcription pre-initiation complex.
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PMID:Histone H1 represses estrogen receptor alpha transcriptional activity by selectively inhibiting receptor-mediated transcription initiation. 1190 41

Cyclin-dependent kinase (CDK)11(p110), formerly known as PITSLRE, is a serine/threonine kinase whose catalytic activity has been associated with transcription and RNA processing. To further evaluate the regulation of CDK11(p110) catalytic activity, interacting proteins were identified by liquid chromatography and tandem mass spectrometry (LC-MS/MS). Following the immunoprecipitation of CDK11(p110) from COS-7 cells, the serine/threonine kinase CK2 was identified by LC-MS/MS. These results were extended through the observation that CDK11(p110) serves as a substrate for CK2 and the identification of a phosphorylation site on CDK11(p110) at Ser227 by LC-MS/MS. To obtain CDK11(p110) devoid of CK2, CDK11(p110) was expressed in High Five insect cells and secreted into the media due to the presence of a honeybee melittin signal sequence encoded at the amino-terminus of CDK11(p110). Recombinant CDK11(p110) was purified from the media and phosphorylation of histone H1 subsequently demonstrated. After demonstrating retention of CDK11(p110) kinase activity, it was evaluated for activity on the carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAP II), but only CK2 was found to phosphorylate the CTD.
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PMID:Cyclin-dependent kinase 11(p110) activity in the absence of CK2. 1464 19

The S143F lamin A/C point mutation causes a phenotype combining features of myopathy and progeria. We demonstrate here that patient dermal fibroblast cells have dysmorphic nuclei containing numerous blebs and lobulations, which progressively accumulate as cells age in culture. The lamin A/C organization is altered, showing intranuclear and nuclear envelope (NE) aggregates and presenting often a honeycomb appearance. Immunofluorescence microscopy showed that nesprin-2 C-terminal isoforms and LAP2alpha were recovered in the cytoplasm, whereas LAP2beta and emerin were unevenly localized along the NE. In addition, the intranuclear organization of acetylated histones, histone H1 and the active form of RNA polymerase II were markedly different in patient cells. A subpopulation of mutant cells, however, expressing the 800 kDa nesprin-2 giant isoform, did not show an overt nuclear phenotype. Ectopic expression of p.S143F lamin A in fibroblasts recapitulates the patient cell phenotype, whereas no effects were observed in p.S143F LMNA keratinocytes, which highly express nesprin-2 giant. Overexpression of the mutant lamin A protein had a more severe impact on the NE of nesprin-2 giant deficient fibroblasts when compared with wild-type. In summary, our results suggest that the p.S143F lamin A mutation affects NE architecture and composition, chromatin organization, gene expression and transcription. Furthermore, our findings implicate a direct involvement of the nesprins in laminopathies and propose nesprin-2 giant as a structural reinforcer at the NE.
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PMID:Nesprin-2 giant safeguards nuclear envelope architecture in LMNA S143F progeria cells. 1788 56


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