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)

The spleen of the ex-hypoxic polycythemic mouse was employed to study the effect of erythropoietin on nuclear RNA polymerase activity. On the basis of ionic strength requirements and sensitivity to the fungal toxin alpha-amanitin, two major forms (I and II) of nuclear RNA polymerase were identified. Within 0.5 h after administration of erythropoietin, at a time when no morphologically identifiable erythroblasts were present in the spleen, there was an increase in the activity of polymerase II. By 2 h, polymerase II activity had declined to control levels. At 3 h, polymerase I activity began to increase, rising to a peak, 88% above control levels, by 12 h. During this period, early erythroblasts began to appear in the spleen. At 12 h, a second increase of similar magnitude occurred in polymerase II activity. Polymerase I activity fell to control levels by 18 h while polymerase II declined more slowly. These data indicate that stimulation of transcription is an early effect of erythropoietin. Multiple forms of RNA polymerase are involved and activation of these is sequential. Nuclear RNA polymerase activity is maximal during the period of early erythroblast proliferation and declines as these cells mature.
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PMID:Sequential activation of splenic nuclear RNA polymerases by erythropoietin. 124 99

We have resolved, by native gel electrophoresis, two intermediates in the transcription of a vaccinia virus early gene by the virus-encoded RNA polymerase. Polymerase holoenzyme containing the vaccinia virus early transcription factor (VETF) forms a complex of VETF bound to the promoter as the first step in a pathway leading to establishment of a committed ternary elongation complex. Formation of the VETF-DNA complex is stimulated by magnesium but is uninfluenced by nucleoside triphosphates. A stable binary complex of RNA polymerase bound to DNA is not detected. Assembly of a gel-stable polymerase-DNA complex depends on conditions permissive for RNA synthesis. Nucleotide omission experiments suggest that at least a tetrameric RNA must be made before a ternary complex is stabilized. RNA analysis indicates that complexes containing nascent transcripts 20 nucleotides long are stable and active. Ternary complex formation requires hydrolyzable ATP. This is consistent with an essential role for the ATPase activity of VETF at a step subsequent to DNA binding, as proposed by Broyles (S. S. Broyles, J. Biol. Chem. 266:15545-15548, 1991). The ternary complex, once formed, is resistant to dissociation by competitor DNA, as well as by salt, Sarkosyl, and heparin. The effects of these inhibitory agents on transcription complex formation suggest that they target different steps in the assembly pathway.
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PMID:Ternary complex formation by vaccinia virus RNA polymerase at an early viral promoter: analysis by native gel electrophoresis. 137 99

In transcriptionally active complexes between RNA polymerase and promoters, the center of the melted region is hyperreactive to the nucleolytic activity of the cuprous complex of 1,10-phenanthroline (OP-Cu). In the first part of this work, using synthetic oligonucleotides and exploiting gel retardation assays, I demonstrate that DNA unpairing is not the only determinant of this hyperreactivity. Polymerase binding is directly implicated, presumably participating in the stabilization of an intermediate required for the cutting. In the second part of the work, I show that, from fine analysis of the nucleolytic pattern of lacUV5 promoter DNA towards OP-Cu and Phe OP-Cu, it is possible to locate polymerase and to characterize its contacts at any time during the early stages of transcription. This analysis provides a description of the passage from the "open complex" to the elongation mode in terms of, first, release of the upstream contacts, and second, loss of sigma subunit. Occupancy of the overlapping promoter, P2, has a positive effect on the escape of polymerase from abortive cycling. The involvement of sigma and beta subunits in the reactivity pattern is discussed with respect to previous cross-linking studies.
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PMID:Passage of RNA polymerase from open complex to elongation mode at the Escherichia coli lacUV5 promoter: nucleolytic hypersensitivity as a probe for complex conformational changes. 142 Jan 67

The components required for specific transcription of ribosomal RNA were isolated from logarithmically growing Acanthamoeba castellanii. The transcription initiation factor fraction, TIF, and RNA polymerase I were extracted from whole cells at 0.35 M KCl. The extract was fractionated with polyethylenimine, then chromatographed on phosphocellulose (P11) which resulted in the separation of TIF from RNA polymerase I. The fractions containing TIF were further chromatographed on DEAE cellulose (DE52), Heparin Affigel, and Matrex green agarose, followed by sedimentation through glycerol gradients. TIF was purified approximately 17,000-fold, and shown to have a native molecular weight of 289 kD, and to bind specifically to rRNA promoter sequences by DNase I footprinting. The addition of homogeneous RNA polymerase I to this complex permitted the initiation of specific transcription in vitro. The phosphocellulose fractions containing RNA polymerase I were chromatographed on DEAE cellulose, Heparin-Sepharose, DEAE-Sephadex, and sedimented through sucrose gradients. Polymerase I was purified to apparent homogeneity with a yield of 8.1% and a specific activity of 315. It contained one fewer subunit than previously reported. DNase I protection experiments demonstrated that in both partially purified and homogeneous fractions, RNA polymerase I was capable of stable binding to the TIF-rDNA complex, and correctly initiating transcription on rDNA templates.
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PMID:Purification of components required for accurate transcription of ribosomal RNA from Acanthamoeba castellanii. 162 Jun 19

It is often assumed that a polymerase moves along the template as it synthesizes RNA. However, a polymerase that tracks along a helical strand will generate a transcript that is entwined about the template. No such interlocking results if the polymerase is immobile and the template moves past it. Therefore we investigated whether immobilization inhibits the RNA polymerase of T7 bacteriophage using a hybrid protein, in which the polymerase is connected through a peptide linker to an immobilizing domain, which in turn was attached through an antibody to protein A covalently linked to plastic beads. Polymerase could be released by cleaving the linker with a protease, factor Xa. Comparison of the activity of the bound and free enzymes showed that immobilization reduced the rate of initiation about fivefold. However, when re-initiation was eliminated by removing excess template, immobilization was found to have little effect on the rate of elongation. Perhaps the untwining problem is sidestepped in vivo by immobilizing the polymerase.
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PMID:Transcription by an immobilized RNA polymerase from bacteriophage T7 and the topology of transcription. 164 25

A polyacrylamide gel assay is used to measure the kinetics of adding a single deoxyribonucleotide onto either a correctly matched or mismatched primer 3' terminus (on M13 template) for all possible DNA base pairs and mispairs using Drosophila melanogaster DNA polymerase alpha (Pol alpha) and avian myeloblastosis virus reverse transcriptase. The reverse transcriptase catalyzes chain extension from transition mispairs (Pur.Pyr and Pyr.Pur, where Pur is purine and Pyr is pyrimidine) more efficiently than polymerase alpha. Reverse transcriptase extends G(primer).T almost 20% as efficiently as it extends A.T, while Pol alpha's G.T extension efficiency is less than 1%. For transversion mispairs (Pur.Pur and Pyr.Pyr), reverse transcriptase extends C.T and T.T with greater efficiency than polymerase alpha, while polymerase alpha is more efficient at extending A.G and G.G mispairs. Reverse transcriptase and polymerase alpha extend the G.G mispair at an efficiency of only 10(-6) and 10(-5), respectively, compared with G.C extension. The extension data for the two polymerases are compared with previously reported nucleotide misinsertion data for the same enzymes (Mendelman, L. V., Boosalis, M. S., Petruska, J., and Goodman, M. F. (1989) J. Biol. Chem. 264, 14415-14423). While the results obtained with reverse transcriptase and Pol alpha differ in detail, some general rules are indicated: (a) Pur.Pyr and Pyr.Pur mispairs, especially G.T and T.G, are easy to insert and even easier to extend; (b) Pyr.Pyr mispairs, especially C.C, are difficult to insert and slightly easier to extend; (c) Pur.Pur mispairs, notably G.G, are harder to extend than to insert. The comparison also shows that reverse transcriptase extends almost all mismatches more efficiently than it forms them, G.G being the only mismatch having a significantly lower efficiency of extension than insertion. Polymerase alpha inserts A.A mismatches most efficiently, but extends them inefficiently, thereby reducing the probability that such transversion mutations will occur in vivo. We show theoretically that when mispaired primers compete with properly matched primers for extension by polymerase, the relative velocities of extension depend on the concentration of the next correct dNTP substrate. The extension velocities depart from Michaelis-Menten kinetics by exhibiting positive cooperativity with respect to substrate concentration.
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PMID:Base mispair extension kinetics. Comparison of DNA polymerase alpha and reverse transcriptase. 168 52

Analysis of porcine transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus (PRCV) mRNA species indicated a deletion in mRNA 3 of PRCV. Polymerase chain reaction (PCR) was used to clone the 5' end of mRNA 3 from PRCV for comparison with the equivalent region in TGEV. Small deletions were observed within and around the PRCV sequence equivalent to the putative open reading frame (ORF) ORF-3a identified in TGEV. The potential RNA polymerase-leader complex binding site (leader RNA binding site), ACTAAAC, found upstream of ORF-3a in TGEV, was absent from the PRCV genome but a potential site was found in the PRCV genome upstream of a gene equivalent to TGEV ORF-3b. PCR analysis, using primers corresponding to sequences within the ORF-3b gene and the leader RNA sequence, confirmed that the leader RNA binding site was upstream of a gene equivalent to TGEV ORF-3b on PRCV mRNA 3 but upstream of ORF-3a on TGEV mRNA 3. The presence of the new leader RNA binding site would be responsible for generating the smaller mRNA 3 species found in PRCV-infected cells.
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PMID:Sequence comparison of the 5' end of mRNA 3 from transmissible gastroenteritis virus and porcine respiratory coronavirus. 184 93

Acanthamoeba rRNA transcription involves the binding of a transcription initiation factor (TIF) to the core promoter of rDNA to form the preinitiation complex. This complex is formed in the absence of RNA polymerase I, and persists for multiple rounds of initiation. Polymerase I next binds to form the initiation complex. This binding is DNA sequence-independent, and is directed by protein-protein contacts with TIF. DNA melting occurs in a separate step. In contrast to most prokaryotic transcription, melting occurs only following nucleotide addition and beta-gamma hydrolysis of ATP is not required as for polymerase II. Growth-dependent regulation of rRNA transcription is accomplished by modification of RNA polymerase I. The inactive form of polymerase (PolE) is unable to bind to the promoter and has altered heat stability. PolE is still active in elongation; thus, the modification affects the polymerase site involved in TIF contact. Modification of a polymerases I and III common subunit has been detected leading to the suggestion that transcription of stable RNAs of the ribosome might be co-regulated by this mechanism.
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PMID:Initiation and regulation mechanisms of ribosomal RNA transcription in the eukaryote Acanthamoeba castellanii. 192 90

A soluble RNA-dependent RNA polymerase was isolated from Nicotiana tabacum plants infected with cucumber mosaic virus (CMV), which has a genome of three positive-strand RNA components, 1, 2, and 3. The purified polymerase contained two virus-encoded polypeptides and one host polypeptide. Polymerase activity was completely dependent on addition of CMV RNA as template, and the products of reaction were single-stranded (ss) RNA and double-stranded (ds) RNA, corresponding to RNAs 1, 2, and 3, and a subgenomic RNA (RNA 4) derived from RNA 3. The ratio of ssRNA to dsRNA was about 5:1, and the ssRNA was shown to be predominantly the positive strand. This demonstrates the complete replication of a eukaryotic virus RNA in vitro by a template-dependent RNA polymerase.
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PMID:Complete replication of a eukaryotic virus RNA in vitro by a purified RNA-dependent RNA polymerase. 220 91

The kinetics of forming all possible single base substitution errors are measured for Drosophila melanogaster DNA polymerase alpha and avian myeloblastosis virus reverse transcriptase. Seventeen sites along bacteriophage M13 DNA are investigated so that effects of nearest neighbor base stacking on misinsertion kinetics can be evaluated. Polymerase alpha appears to be more error prone than reverse transcriptase. Polymerase alpha forms transversion mispairs at rates comparable to transition mispairs with two exceptions; A.A and C.C are formed with significantly higher and lower efficiencies, respectively. Reverse transcriptase forms transversions with lower efficiencies than transitions, especially low being A.G, G.G, and C.C. For both enzymes, misinsertion frequencies vary typically by 10-fold for the same mispair in different locations. Misinsertion frequency can be expressed as a product of two components, one based on Km and the other on Vmax. DNA polymerase alpha appears to use primarily Km discrimination (100-5000-fold) to achieve insertion fidelity while reverse transcriptase shows a greater balance between Km and Vmax discrimination. Nearest-neighbor base stacking interactions appear to have opposite effects on the two discrimination components. The 5'-nearest neighbor influence on Km is greater for correct insertions than for incorrect, while the influence on Vmax is greater for the incorrect base. Target sites that have pyrimidine as the 5'-nearest neighbor to incoming nucleotides show a higher than average misinsertion component based on Km, but a lower than average component based on Vmax. Conversely, target sites with nearest neighbor purines have a higher than average Vmax component. These results imply that nucleotide misinsertion "hot spots" will occur next to pyrimidines when Km discrimination is dominant and next to purines when Vmax discrimination is dominant. When Vmax and Km discrimination components have similar magnitudes, nearest neighbor effects tend to cancel thereby reducing the effects of base stacking on insertion error rates.
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PMID:Nearest neighbor influences on DNA polymerase insertion fidelity. 247 45


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