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)

Replication complexes were isolated from alfalfa mosaic virus-infected tobacco protoplasts. Most of the RNA-synthesizing activity appears to colocalize with the intact chloroplasts upon sucrose-gradient centrifugation of cellular homogenates. Further analysis of these replication complexes showed that the enzyme is strongly associated with the outside of the chloroplasts, the endogenous template being well protected against ribonuclease action. RNA polymerase activity is sensitive to protease treatment of intact chloroplast fraction showing that an essential part of the enzyme complex is facing the in vitro medium, and probably the cytosol in vivo.
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PMID:Localization and biochemical characterization of alfalfa mosaic virus replication complexes. 850 93

Elongation factor SII is required to increase the efficiency of transcription by RNA polymerase II through intrinsic arrest sites. RNA polymerase II ternary complexes exhibit a ribonuclease activity in the presence of SII, truncating nascent transcripts in a 3'-->5' direction. We show here that transcript cleavage is an obligatory step in re-establishing the elongation competency of complexes that have become blocked in elongation at an intrinsic arrest site. SII-facilitated transcript cleavage by these arrested complexes released 7-14 nucleotide RNA fragments. In contrast, SII-facilitated transcript cleavage by elongation competent complexes, which are stalled because of the absence of a nucleoside triphosphate from the reaction mixture, occurred primarily in dinucleotide increments. We can partially recreate the arrested phenotype and the preference for the large cleavage increment by stalling ternary complexes such that the 3'-end of the transcript contains consecutive U residues, which mimics the sequence of the 3'-ends of transcripts in arrested complexes.
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PMID:The increment of SII-facilitated transcript cleavage varies dramatically between elongation competent and incompetent RNA polymerase II ternary complexes. 850 21

Gel purification of radioactive riboprobes enhances the quality of the ribonuclease protection assay. A simple and effective method for riboprobe purification is described. The method uses acrylamide gels in plastic tubes to achieve electrophoretic separation of the RNA polymerase products.
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PMID:Simplified riboprobe purification using translucent straws as gel tubes. 867 36

RNA polymerases encounter specific DNA sites at which RNA chain elongation takes place in the absence of enzyme translocation in a process called discontinuous elongation. For RNA polymerase II, at least some of these sequences also provoke transcriptional arrest where renewed RNA polymerization requires elongation factor SII. Recent elongation models suggest the occupancy of a site within RNA polymerase that accommodates nascent RNA during discontinuous elongation. Here we have probed the extent of nascent RNA extruded from RNA polymerase II as it approaches, encounters, and departs an arrest site. Just upstream of an arrest site, 17-19 nucleotides of the RNA 3'-end are protected from exhaustive digestion by exogenous ribonuclease probes. As RNA is elongated to the arrest site, the enzyme does not translocate and the protected RNA becomes correspondingly larger, up to 27 nucleotides in length. After the enzyme passes the arrest site, the protected RNA is again the 18-nucleotide species typical of an elongation-competent complex. These findings identify an extended RNA product groove in arrested RNA polymerase II that is probably identical to that emptied during SII-activated RNA cleavage, a process required for the resumption of elongation. Unlike Escherichia coli RNA polymerase at a terminator, arrested RNA polymerase II does not release its RNA but can reestablish the normal elongation mode downstream of an arrest site. Discontinuous elongation probably represents a structural change that precedes, but may not be sufficient for, arrest by RNA polymerase II.
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PMID:Increased accommodation of nascent RNA in a product site on RNA polymerase II during arrest. 869 22

We have investigated the endonuclease activity of the influenza A virus RNA polymerase in an in vitro assay with an artificial influenza-like mRNA containing a cap structure at its 5' terminus, followed by a 10 nt beta-globin mRNA sequence, and the 5' and 3' conserved termini of a truncated nucleoprotein (NP) cRNA influenza sequence. Results showed that partially purified virion ribonucleoprotein complexes (RNPs) and micrococcal nuclease treated RNPs cleaved the artificial influenza-like mRNA substrate specifically at positions near the 5' terminus to generate capped 14 and 15 nucleotide long RNA fragments which subsequently served as primers to initiate transcription. The endonuclease activity was completely blocked by addition of cap analog and competitively inhibited by added globin mRNA. Furthermore, an in vitro reconstituted influenza RNA transcription reaction containing a truncated NP vRNA as template, micrococcal nuclease treated RNPs and globin mRNA as primer, synthesized capped and uncapped full length (+) sense products. Enzyme kinetics showed that capped RNA was made earlier in the reaction; it reached a peak at 120 min and then declined. However, uncapped cRNA synthesis appeared later and remained as the dominant product later in the reaction. The nature of these products was confirmed by ribonuclease protection assays and by primer extension.
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PMID:Influenza A virus RNA-dependent RNA polymerase cleaves influenza mRNA in vitro. 880 82

Fidelity of DNA and protein synthesis is regulated by a proofreading mechanism but function of a similar mechanism during RNA synthesis has not been demonstrated. Analysis of transcriptional fidelity and its control has been hampered by the necessity to employ complex DNA templates requiring either a promoter and initiation factors or 3'-extended templates. To circumvent this difficulty, we have created an RNA-DNA dumbbell template that can be recognized as a template-primer and extended by RNA polymerase II. By employing this system, we demonstrate that RNA polymerase II can misincorporate a nucleotide and carry out template-dependent elongation at the mispaired end. The transcripts containing misincorporated residues can be cleaved by the very slow 3'-->5' ribonuclease activity of the RNA polymerase II, but enhancement of this activity by the elongation factor TFIIS generates RNA with a high degree of fidelity. This enhanced preferential cleavage of misincorporated transcripts suggests an important role for TFIIS in maintaining transcriptional fidelity.
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PMID:Fidelity of RNA polymerase II transcription controlled by elongation factor TFIIS. 894 93

RNA polymerase II contains a ribonuclease activity which is stimulated by the transcription elongation factor SII. This nuclease shortens the nascent RNA and facilitates relief of transcriptional arrest by allowing the enzyme to make multiple attempts to read through an obstacle to transcription. The catalytic center of this ribonuclease is unknown, although a region of the enzyme's second largest subunit shares local sequence similarly with barnase and other bacterial ribonucleases. To test the role of the barnase homology region in SII-activated cleavage, we engineered a single amino acid change in the Saccharomyces cerevisiae enzyme at a position homologous to a catalytic residue of barnase (Glu-371) and has been suggested as a participant in active site chemistry of RNA polymerase II. We purified RNA polymerase II from mutant yeast and assayed its ability to cleave and re-extend the nascent RNA following SII treatment. We find no defects in this function of the mutant enzyme, suggesting that the barnase homology region does not represent the active site of the SII-activated nuclease. These mutant yeast cells were also resistant to mycophenolic acid, which slows the growth of some yeast mutants bearing elongation defective RNA polymerase II or mutant elongation factor SII.
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PMID:Glutamic acid-371 of the barnase homology domain in RNA polymerase II is not required for SII-activated RNA cleavage. 903 12

The rpoS gene of Escherichia coli encodes an alternative sigma factor of RNA polymerase sigma38 (or sigma(s)) that is required for transcription of katE encoding catalase HPII. The transcription start site of the single katE transcript identified by ribonuclease protection has been determined by primer extension analysis to be either 53 or 54 bp (depending on the strain used) upstream of the open reading frame. A series of promoter fragments were constructed and fused to lacZ to confirm the start site location. A - 10 sequence similar to that found in other sigma70- and sigma38-dependent E. coli promoters was identified 8 or 7 bp upstream of the start site but a sigma70-dependent -35 sequence was not evident.
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PMID:Identification and analysis of the rpoS-dependent promoter of katE, encoding catalase HPII in Escherichia coli. 919 47

Human eosinophils contain a number of granule proteins for which specific physiological roles remain unclear. The combined ribonucleolytic and membrane disruptive properties of the eosinophil-derived neurotoxin and eosinophil cationic protein, respectively, suggest the possibility that eosinophils might participate in host defense against enveloped single-stranded RNA viruses. To test this hypothesis, stocks of a replication-defective retrovirus encoding the reporter gene beta-galactosidase were pretreated with isolated human eosinophils, then used to transduce human erythroleukemia (K-562) target cells. Histochemical staining for beta-galactosidase activity was used to detect and quantitate the transduced cells. Co-incubation of retrovirus with eosinophils (0.4 x 10[6]/mL) before target cell transduction resulted in a marked decrease in transduction efficiency corresponding to an approximately 20-fold dilution of viral stock (P < 0.01), an effect that was directly proportional to the concentration of eosinophils, and that was reversed in the presence of ribonuclease inhibitor. Reverse transcriptase-polymerase chain reaction analysis demonstrated loss of the retroviral RNA genome as a result of eosinophil pretreatment, indicating that eosinophils are capable of mediating direct ribonucleolytic destruction of the isolated retroviral particles. Our results demonstrate that eosinophils function as effective anti-retroviral agents in vitro via the actions of their secreted ribonucleases, and suggest that eosinophils may represent an unrecognized arm of host defense against enveloped single-stranded RNA viral pathogens.
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PMID:Eosinophils inhibit retroviral transduction of human target cells by a ribonuclease-dependent mechanism. 930 75

While there is considerable evidence for phosphate (Pi) reabsorption in the distal tubule, Pi transport and its regulation have not been well characterized in this segment of the nephron. In the present study, we examined Na+-dependent Pi transport in immortalized mouse distal convoluted tubule (MDCT) cells. Pi uptake by MDCT cells is Na+-dependent and, under initial rate conditions, is inhibited by phosphonoformic acid (41 +/- 3% of control), a competitive inhibitor of Na+-Pi cotransport. The transport system has a high affinity for Pi (Km = 0.46 mM) and is stimulated by lowering the extracellular pH from 7.4 to 6.4 and inhibited by raising the pH from 7.4 to 8.4. Exposure to Pi-free medium for 21 h increased Na+-Pi cotransport from 2.1 to 5.5 nmol/mg of protein/5 minutes (p < 0.05) while parathyroid hormone, forskolin, and phorbol 12-myristate 13-acetate failed to alter Pi uptake in MDCT cells. Reverse transcriptase polymerase chain reaction of MDCT cell RNA provided evidence for the expression of the Npt1 but not the Npt2 Na+-Pi cotransporter gene. However, preincubation of MDCT cells with Npt1 antisense oligonucleotide led to only 20% inhibition of Na+-Pi cotransport, suggesting that other Na+-Pi cotransporters are operative in MDCT cells. Indeed, we showed, by ribonuclease protection assay, that MDCT cells express the ubiquitous cell surface receptors for gibbon ape leukemia virus (Glvr-1) and amphoteric murine retrovirus (Ram-1) that also function as Na+-Pi cotransporters. In summary, we demonstrate that the pH dependence and regulation of Na+-Pi cotransport in MDCT cells is distinct from that in the proximal tubule and suggest that different gene products mediate Na+-Pi cotransport in the proximal and distal segments of the nephron.
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PMID:Na+ -phosphate cotransport in mouse distal convoluted tubule cells: evidence for Glvr-1 and Ram-1 gene expression. 955 59


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