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)

Evidence is presented that the activation of the RNA polymerase sigma factor sigma(W) in Bacillus subtilis by regulated intramembrane proteolysis is governed by a novel, membrane-embedded protease. The sigma(W) factor is activated by proteolytic destruction of the membrane-bound anti-sigma(W) factor RsiW in response to antimicrobial peptides and other agents that damage the cell envelope. RsiW is destroyed by successive proteolytic events known as Site-1 and Site-2 cleavage. Site-2 cleavage is mediated by a member of the SpoIVFB-S2P family of intramembrane-acting metalloproteases, but the protease responsible for Site-1 cleavage was unknown. We have identified a previously uncharacterized, multipass membrane protein called PrsW (annotated YpdC) that is both necessary and sufficient (when artificially produced in an unrelated host bacterium) for Site-1 cleavage of RsiW. PrsW is a member of a widespread family of membrane proteins that includes at least one previously known protease. We identify residues important for proteolysis and a cluster of acidic residues involved in sensing antimicrobial peptides and cell envelope stress.
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PMID:Evidence for a novel protease governing regulated intramembrane proteolysis and resistance to antimicrobial peptides in Bacillus subtilis. 1681

Activation of the luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor (LHR) in cultured hypothalamic cells and immortalized GnRH (gonadotropin-releasing hormone) neurons (GT1-7 cells) transiently stimulates and subsequently inhibits cAMP production and pulsatile GnRH release. The marked and delayed impairment of cAMP signaling and episodic GnRH release in GT1-7 cells is prevented by pertussis toxin (PTX). This, and the LH-induced release of membrane-bound Galpha(s) and Galpha(i3) subunits, are indicative of differential G protein coupling to the LHR. Action potential (AP) firing in identified GnRH neurons also initially increased and then progressively decreased during LH treatment. The inhibitory action of LH on AP firing was also prevented by PTX. Reverse transcriptase-PCR analysis of GT1-7 neurons revealed the expression of G protein-gated inwardly rectifying potassium (GIRK) channels in these cells. The LH-induced currents were inhibited by PTX and were identified as GIRK currents. These responses indicate that agonist stimulation of endogenous LHR expressed in GnRH neurons activates GIRK channels, leading to suppression of membrane excitability and inhibition of AP firing. These findings demonstrate that regulation of GIRK channel function is a dominant factor in gonadotropin-induced abolition of pulsatile GnRH release. Furthermore, this mechanism could contribute to the suppression of pituitary function during pregnancy.
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PMID:Essential role of G protein-gated inwardly rectifying potassium channels in gonadotropin-induced regulation of GnRH neuronal firing and pulsatile neurosecretion. 1682 87

Cereal yellow dwarf virus (CYDV) RNA has a 5'-terminal genome-linked protein (VPg). We have expressed the VPg region of the CYDV genome in bacteria and used the purified protein (bVPg) to raise an antiserum which was able to detect free VPg in extracts of CYDV-infected oat plants. A template-dependent RNA-dependent RNA polymerase (RdRp) has been produced from a CYDV membrane-bound RNA polymerase by treatment with BAL 31 nuclease. The RdRp was template specific, being able to utilize templates from CYDV plus- and minus-strand RNAs but not those of three unrelated viruses, Red clover necrotic mosaic virus, Cucumber mosaic virus, and Tobacco mosaic virus. RNA synthesis catalyzed by the RdRp required a 3'-terminal GU sequence and the presence of bVPg. Additionally, synthesis of minus-strand RNA on a plus-strand RNA template required the presence of a putative stem-loop structure near the 3' terminus of CYDV RNA. The base-paired stem, a single-nucleotide (A) bulge in the stem, and the sequence of a tetraloop were all required for the template activity. Evidence was produced showing that minus-strand synthesis in vitro was initiated by priming by bVPg at the 3' end of the template. The data are consistent with a model in which the RdRp binds to the stem-loop structure which positions the active site to recognize the 3'-terminal GU sequence for initiation of RNA synthesis by the addition of an A residue to VPg.
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PMID:In vitro synthesis of minus-strand RNA by an isolated cereal yellow dwarf virus RNA-dependent RNA polymerase requires VPg and a stem-loop structure at the 3' end of the virus RNA. 1692 57

Replication of the plus-stranded RNA genome of hepatitis C virus (HCV) occurs in a membrane-bound replication complex consisting of viral and cellular proteins and viral RNA. NS5B, the RNA polymerase of HCV, is anchored to the membranes via a C-terminal 20-amino-acid-long hydrophobic domain, which is flanked on each side by a highly conserved positively charged arginine. Using a genotype 1b subgenomic replicon (V. Lohmann, F. Korner, J. O. Koch, U. Herian, L. Theilmann, and R. Bartensclager, Science 285:110-113, 1999), we determined the effect of mutations of some highly conserved residues in this domain. The replacement of arginine 570 with alanine completely abolished the colony-forming ability by the replicon, while a R591A change was found to be highly detrimental to replication, viability, and membrane binding by the mutant NS5B protein. Mutations of two other highly conserved amino acids (L588A and P589A) reduced but did not eliminate colony formation. It was of interest, if specific amino acid residues play a role in membrane anchoring of NS5B and replication, to determine whether a complete exchange of the NS5B hydrophobic domain with a domain totally unrelated to NS5B would ablate replication. We selected the 22-amino-acid-long hydrophobic domain of poliovirus polypeptide 3A that is known to adopt a transmembrane configuration, thereby anchoring 3A to membranes. Surprisingly, either partial or full replacement of the NS5B hydrophobic domain with the anchor sequences of poliovirus polypeptide 3A resulted in the replication of replicons whose colony-forming abilities were reduced compared to that of the wild-type replicon. Upon continued passage of the replicon in Huh-7 cells in the presence of neomycin, the replication efficiency of the replicon increased. However, the sequence of the poliovirus polypeptide 3A hydrophobic domain, in the context of the subgenomic HCV replicon, was stably maintained throughout 40 passages. Our results suggest that anchoring NS5B to membranes is necessary but that the amino acid sequence of the anchor per se does not require HCV origin. This suggests that specific interactions between the NS5B hydrophobic domain and other membrane-bound factors may not play a decisive role in HCV replication.
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PMID:The C-terminal hydrophobic domain of hepatitis C virus RNA polymerase NS5B can be replaced with a heterologous domain of poliovirus protein 3A. 1697 30

Vascular endothelial growth factor (VEGF) plays a critical role during normal embryonic angiogenesis and also in the pathological angiogenesis that occurs in a number of diseases, including cancer. K562 human leukemia cells overexpress VEGF, with a shift in isoform production from membrane-bound VEGF189 to the more soluble VEGF165. In the present study, three 19 bp reverse repeated motifs targeting exons 5 and 7 boundary of VEGF165 gene sequence with 9 bp spacer were synthesized and cloned into eukaryotic expression plasmid pGenesil-1 containing U6 shRNA promoter and termination signal of RNA polymerase. The recombinant plasmids pGenesil-VR1, pGenesil-VR2, pGenesil-VR3 and pGenesil-con (plasmid containing random DNA fragment) were transfected into K562 cells, respectively, through lipofectamine reagent. A vector-based small interfering RNA(SiRNA) inhibited VEGF165 mRNA expression by 72% and protein production by 67% in K562 cells. Human microvascular endothelial cell migration induced by conditioned medium from VEGFsi-transfected K562 cells was significantly less than that induced by conditioned medium from K562 cells and control vector-transfected K562 cells. Furthermore, the VEGF shRNA dramatically suppressed tumor angiogenesis and tumor growth in a K562 s.c. xenograft model. Vessel density as assessed by vWF immunohistochemical analysis was also decreased. This strategy provides a novel tool to study the function of various VEGF isoforms and may contribute to VEGF-specific treatment in cancer.
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PMID:Vector-based RNAi approach to isoform-specific downregulation of vascular endothelial growth factor (VEGF)165 expression in human leukemia cells. 1703 51

Replication of poliovirus RNA takes place on the cytoplasmic surface of membranous vesicles that form after infection of the host cell. It is generally accepted that RNA polymerase 3D(pol) interacts with membranes in a complex with viral protein 3AB, which binds to membranes by means of a hydrophobic anchor sequence that is located near the C-terminus of the 3A domain. In this study, we used fluorescence and fluorescence quenching methods to define the topography of the anchor sequence in the context of 3A and 3AB proteins inserted in model membranes. Mutants with a single tryptophan near the center of the anchor sequence but lacking Trp elsewhere in 3A/3AB were constructed which, after the emergence of suppressor mutations, replicated well in HeLa cells. When a peptide containing the mutant anchor sequence was incorporated in model membrane vesicles, measurements of Trp depth within the lipid bilayer indicated formation of a transmembrane topography. However, rather than the 22-residue length predicted from hydrophobicity considerations, the transmembrane segment had an effective length of 16 residues, such that Gln64 likely formed the N-terminal boundary. Analogous experiments using full-length proteins bound to preformed model membrane vesicles showed that the anchor sequence formed a mixture of transmembrane and nontransmembrane topographies in the 3A protein but adopted only the nontransmembrane configuration in the context of 3AB protein. Studies of the function of 3A/3AB inserted into model membrane vesicles showed that membrane-bound 3AB is highly efficient in stimulating the activity of 3D(pol) in vitro while membrane-bound 3A totally lacks this activity. Moreover, in vitro uridylylation reactions showed that membrane-bound 3AB is not a substrate for 3D(pol), but free VPg released by cleavage of 3AB with proteinase 3CD(pro) could be uridylylated.
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PMID:Membrane topography of the hydrophobic anchor sequence of poliovirus 3A and 3AB proteins and the functional effect of 3A/3AB membrane association upon RNA replication. 1741 22

B cells produce Ig H chain (IgH) mRNA and protein, primarily of the membrane-bound specific form. Plasma cells produce 20- to 50-fold higher amounts of IgH mRNA, most processed to the secretory specific form; this shift is mediated by substantial changes in RNA processing but only a small increase in IgH transcription rate. We investigated RNA polymerase II (RNAP-II) loading and phosphorylation of its C-terminal domain (CTD) on the IgG2a H chain gene, comparing two mouse cell lines representing B (A20) and plasma cells (AxJ) that express the identical H chain gene whose RNA is processed in different ways. Using chromatin immunoprecipitation and real-time PCR, we detected increased RNAP-II and Ser-2 and Ser-5 phosphorylation of RNAP-II CTD close to the IgH promoter in plasma cells. We detected increased association of several 3' end-processing factors, ELL2 and PC4, at the 5' end of the IgH gene in AxJ as compared with A20 cells. Polymerase progress and factor associations were inhibited by 5,6-dichlorobenzimidazole riboside, a drug that interferes with the addition of the Ser-2 to the CTD of RNAP-II. Taken together, these data indicate a role for CTD phosphorylation and polyadenylation/ELL2/PC4 factor loading on the polymerase in the choice of the secretory poly(A) site for the IgH gene.
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PMID:Increased phosphorylation of the carboxyl-terminal domain of RNA polymerase II and loading of polyadenylation and cotranscriptional factors contribute to regulation of the ig heavy chain mRNA in plasma cells. 1802 12

RNA-dependent RNA polymerase activity and protein synthesis in brome mosaic virus (BMV)-infected and uninfected protoplasts were investigated during the course of viral replication. In protoplast homogenates, the membrane-bound RNA polymerase activity resistant to actinomycin D was enhanced by BMV infection up to 30-fold that found in mock-inoculated protoplasts. The activity was first detected 7 to 8 hr postinfection. It reached a maximum at around 30 hr postinfection, then decreased gradually while virus and the viral antigen in the protoplasts, as measured by infectivity and fluorescent antibody staining, respectively, continued to increase. The bound enzyme activity was not enhanced by inoculation with BMV RNA 3 and/or RNA 4. Four proteins with molecular weights of 120, 110, 36, and 19.5 x 103 were observed in BMV-infected protoplasts from three plant species (a systemic host, a local lesion host, and a nonhost of BMV).
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PMID:RNA polymerase activity and protein synthesis in brome mosaic virus-infected protoplasts. 1863 24

A membrane-bound RNA-dependent RNA polymerase from Cowpea mosaic virus (CPMV)-infected cowpea leaves (Vigna unguiculata) has been purified 15,000-fold by DEAE-Sepharose CL-6B chromatography, affinity chromatography on poly(U)-Sepharose 4B, and glycerol gradient centrifugation. Particularly, poly(U)-Sepharose 4B chromatography was a very efficient purification step and, in addition, achieved the separation of a host-encoded terminal uridylyl transferase activity from the RNA polymerase activity. On glycerol gradient centrifugation, the polymerase activity sedimented as a homogeneous peak with a rate corresponding to a molecular weight of 120,000. Analysis of the protein composition of the gradient fractions revealed that only one polypeptide with a molecular weight of 130,000 cosedimented with the polymerase activity, suggesting a monomeric enzyme. The most purified enzyme preparations from CPMV infected leaves did not contain polypeptides encoded by RNA from CPMV B-component which presumably carries functions essential for CPMV replication. Using the same purification procedure, an RNA-dependent RNA polymerase has also been purified from mock-inoculated leaves, which appeared to be identical to the RNA polymerase from infected leaves. This host enzyme was strongly stimulated in cowpea leaves infected with CPMV. The role, if any, of the RNA-dependent RNA polymerase from cowpea leaves in CPMV-RNA replication is discussed in view of the recent evidence for virus encoded functions involved in CPMV multiplication.
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PMID:Purification of a host-encoded RNA-dependent RNA polymerase from cowpea mosaic virus-infected cowpea leaves. 1863 12

The hnRNP C heterotetramer [(C1(3))C2] binds RNA polymerase II transcripts in the nucleus, along with other proteins of the core hnRNP complex, and plays an important role in mRNA biogenesis and transport. Infection of HeLa cells with poliovirus causes hnRNP C to re-localize from the nucleus, where it is normally retained during interphase, to the cytoplasm. We have proposed that in the cytoplasm, the protein isoforms of hnRNP C participate in the recognition of viral specific RNAs by the poliovirus replication proteins and/or in the assembly of membrane-bound RNA replication complexes. In SK-OV-3 cells, which express reduced levels of hnRNP C compared to HeLa cells or 293 cells, the kinetics of poliovirus replication are delayed. hnRNP C is also re-localized from the nucleus to the cytoplasm in SK-OV-3 cells infected with poliovirus. Increased expression of hnRNP C in SK-OV-3 cells by transient transfection increases the rate of virus production and overall yield over that seen in mock-transfected cells. We propose that hnRNP C interacts with poliovirus RNA and replication proteins to increase the efficiency of viral genomic RNA synthesis.
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PMID:Delayed kinetics of poliovirus RNA synthesis in a human cell line with reduced levels of hnRNP C proteins. 2018 23


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