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)

Deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase, EC 3.6.1.23) catalyzes the hydrolysis of dUTP to dUMP and pyrophosphate, and plays important roles in nucleotide metabolism and DNA replication. The dUTPase gene of the retrovirus equine infectious anemia virus (EIAV) was cloned and overexpressed in Escherichia coli using the T7 RNA polymerase expression system. The recombinant vector (pET-3a/EDU), constructed by mutagenic PCR, was transformed into E. coli BL21 (DE3) pLysS cells, resulting in expression of EIAV dUTPase at about 40% of the extracted protein. This level of overproduction is very high compared to previous reports on heterologous expression of dUTPases in E. coli. A one-step purification procedure using phosphocellulose chromatography results in a homogeneous preparation of the enzyme in a yield of 45 mg liter-1 of bacterial culture. The purified EIAV dUTPase, run on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis, shows an apparent molecular mass of 15.1 kDa in accordance with the gene structure. The isoelectric point (pI) was determined to 5.6. Gel filtration under nondenaturating conditions gives a retention volume corresponding to a molecular mass of 40.6 kDa, suggesting a trimeric organization of the enzyme. The amino acid composition and amino-terminal sequence of the recombinant dUTPase are in agreement with predictions from the DNA sequence.
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PMID:dUTPase from the retrovirus equine infectious anemia virus: high-level expression in Escherichia coli and purification. 766 76

In Klebsiella pneumoniae, transcription of all nif (nitrogen fixation) operons except the regulatory nifLA operon itself is regulated by the proteins NifA and NifL. NifA, an enhancer-binding protein, activates transcription by RNA polymerase containing the alternative sigma factor sigma 54. The central catalytic domain of NifA is sufficient for transcriptional activation, which can occur from solution. In vivo, NifL antagonizes the action of NifA in the presence of molecular oxygen or combined nitrogen. Inhibition has also been shown in vitro, but it was not responsive to environmental signals. Assuming a two-domain structure of NifL, we localized inhibition by NifL to its carboxy (C)-terminal domain, which is more soluble than the intact protein. The first line of evidence for this is that internal deletions of NifL containing an intact C-terminal domain were able to inhibit transcriptional activation by NifA in a coupled transcription-translation system. The second line of evidence is that the isolated C-terminal domain of NifL (assayed as a fusion to the soluble maltose-binding protein [MBP]) was sufficient to inhibit transcriptional activation by the central domain of NifA in a purified transcription system. The final line of evidence is that an MBP fusion to the C-terminal domain of NifL inhibited transcriptional activation by NifA in vivo. On the basis of these data, we postulate that the inhibitory function of NifL lies in its C-terminal domain and hence infer that this domain is responsible for interaction with NifA. Gel filtration experiments with MBP-NifL fusion derivatives lacking portions of the N- or C-terminal domain of the protein revealed that the C-terminal domain is the most soluble part of NifL. Up to 50% of two MBP-NifL truncations containing only the C-terminal domain appeared to be in a defined dimeric state.
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PMID:The C-terminal domain of NifL is sufficient to inhibit NifA activity. 766 87

Treatment of bacteriophage T7 with methyl methanesulfonate perturbed phage-specific genetic expression in both repair-proficient and repair-deficient Escherichia coli cells. In wild-type cells (AB1157), the time course of protein synthesis was slowed down but an entire complement of phage proteins was synthesized. In cells (BK2114, tag-) unable to repair 3-methyladenine, the toxic lesion produced by methyl methanesulfonate, alkylated phage produced only early (class I) proteins. These results suggested that late transcription was inhibited in infected tag- cells. These cells were shown to contain a significant amount of active T7 RNA polymerase, a class I protein. Thus, the cause of inhibition appeared to be the inability of T7 RNA polymerase to use unrepaired DNA as template. In vitro transcription assays with alkylated T7 DNA as template supported this proposal. T7 RNA polymerase proved to be very sensitive to the presence of alkylation lesions. In addition, the phage enzyme was much more sensitive to these lesions than was its bacterial counterpart, E. coli RNA polymerase. These results suggest that 3-methyladenine exerts its toxic action, in the T7 system, at the level of transcription by T7 RNA polymerase. To further characterize the reduced activity of the T7 enzyme, an in vitro transcription assay using linearized plasmid DNA with one T7 promoter was devised. Gel electrophoresis revealed that only one transcript of well-defined length was synthesized by T7 RNA polymerase on this template. Alkylation of the template did not alter the size of the transcript produced. Simultaneous measurement of chain initiation and chain elongation confirmed this result by showing that both steps were reduced to the same extent by alkylation of template DNA. Thus T7 RNA polymerase does not appear to be blocked by 3-methyladenine. Rather the lesion must hinder translocation of T7 RNA polymerase along the DNA template during chain elongation.
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PMID:Mechanism of toxicity of 3-methyladenine for bacteriophage T7. 769 68

We have purified, sequenced, and prepared a synthetic clone of a small (60-nucleotide) RNA molecule from the yeast Saccharomyces cerevisiae that had previously been isolated on the basis of its ability to selectively block the translation of poliovirus mRNA. RNA derived from the clone by transcription with T7 RNA polymerase appears to block translation initiation by internal ribosome entry (cap independent) but does not significantly affect cap-dependent translation. Deletion analysis of the poliovirus 5'-untranslated region (5'-UTR) has shown that yeast inhibitor RNA (I-RNA) requires internal ribosome entry site sequences to inhibit the translation of poliovirus RNA in vitro. Using a bicistronic RNA construct, we show that I-RNA preferentially inhibits translation by internal ribosome entry. Gel retardation and UV cross-linking studies demonstrate that I-RNA specifically binds proteins which interact with RNA secondary structures within the poliovirus 5'-UTR presumably involved in internal initiation. Specifically, purified I-RNA competes with virus RNA structures within the 5'-UTR which bind a cellular protein with an approximate molecular mass of 52 kDa. Finally, when transfected into HeLa cells, I-RNA efficiently inhibits the replication of poliovirus RNA presumably by inhibiting translation of the input virus RNA.
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PMID:A small yeast RNA selectively inhibits internal initiation of translation programmed by poliovirus RNA: specific interaction with cellular proteins that bind to the viral 5'-untranslated region. 793 2

The gene 8 product of SA11 rotavirus, NS35 (NSP2), is a nonspecific RNA-binding protein that accumulates in cytoplasmic inclusions (viroplasms) and is required for genome replication. To gain additional information on the role of NS35 in virus replication, lysates of simian rotavirus SA11-infected cells were treated with the thio-cleavable crosslinking agent, dithiobis(succinimidyl propionate) (DSP). Gel electrophoresis of NS35-specific immunoprecipitates recovered from the crosslinked lysates indicated that infected cells contained NS35 multimers, the largest consisting of four or more molecules of the protein. Sedimentation analysis of NS35 expressed in rabbit reticulocyte lysates by cell-free translation and in vTF7-3-infected cells by transfection with a gene 8-containing transcription vector showed that NS35 assembles into multimers of approximately 10S and that the formation of the multimers does not require other viral proteins. The 10S multimers were also detected in rotavirus-infected cells, providing evidence that they function in virus replication. The lack of RNase sensitivity indicates that the 10S multimers probably lack an RNA component. However, by an NS35-specific RNA capture assay, the multimers were shown to possess the RNA-binding activity previously demonstrated for NS35. Despite its ability to multimerize and bind RNA, indirect immunofluorescence assays showed that when transiently expressed in cells, NS35 alone is not sufficient to induce the formation of viroplasms. DSP-crosslinking of infected cell lysates and immunoprecipitation also revealed that NS35 interacts with the putative viral RNA polymerase VP1. Analysis of cytoplasmic extracts resolved by sedimentation on glycerol gradients suggested that the VP1-NS35 complexes are soluble and RNA-free. Complexes formed from NS35 multimers, VP1, and viral messenger RNA may function to coordinate RNA packaging and the assembly of viral cores.
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PMID:The rotavirus RNA-binding protein NS35 (NSP2) forms 10S multimers and interacts with the viral RNA polymerase. 803 Feb 43

The Klebsiella aerogenes hutUH operon is preceded by a promoter region, hut(P), that contains two divergent promoters (hutUp and Pc) which overlap and are alternately expressed. In the absence of the catabolite gene activator protein-cyclic AMP (CAP-cAMP) complex, Pc is predominantly expressed while hutUp is largely repressed. CAP-cAMP has the dual effect of repressing transcription from Pc while simultaneously activating transcription from hutUp. DNA deletion mutations in this region were used to identify DNA sequences required for transcription of these two promoters. We showed that inactivation of Pc by DNA deletion did not result in activation of hutUp in vitro or in vivo. In addition, Escherichia coli CAP mutants that are known to bind and bend DNA normally but are unable to activate various CAP-dependent promoters were also unable to activate hutUp in vivo. These results invalidate an indirect activation model by which CAP-mediated repression of Pc in itself would led to activation of hutUp. Gel retardation asays with various deletion mutations of hut(P) and DNase I protection analyses revealed a high-affinity CAP binding site (CAP site 1) centered at -81.5 relative to the hutUp start of transcription and a second low-affinity CAP site (CAP site 2) centered at about -41.5. CAP site 1 is essential for activation of hutUp. Although CAP site 2 by itself is unable to activate hutUp in vivo under catabolite-activating conditions, it appears to be required for maximal transcription from a site centered at -41.5, does not activate hutUp suggests that the role of CAP-cAMP at the weaker CAP site may be different from that of other promoters containing a similarly positioned site. We propose that CAP directly stimulates the activity of RNA polymerase at hutUp and that this reaction is completely dependent on a naturally occurring CAP site centered at -81.5 and also involves a second CAP site centered at about -41.5 for maximal activation.
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PMID:Roles of catabolite activator protein sites centered at -81.5 and -41.5 in the activation of the Klebsiella aerogenes histidine utilization operon hutUH. 807 Dec 30

The interactions of transcription inhibitors with the open complex composed of Escherichia coli RNA polymerase and the lacUV5 promoter have been studied using gel retardation, the chemical nuclease activity of the cuprous complexes of 1,10-phenanthroline (OP) and its derivatives, and steady-state kinetics. Gel retardation shows that two inhibitors, the 2:1 2,9-dimethyl-1,10-phenanthroline-cuprous complex [(2,9-Me2OP)2Cu+] and rifampicin, bind stably to the open-complex. (2,9-Me2OP)2Cu+ blocks scission by the chemical nuclease by interfering with the binding of its redox-active isosteres. Rifampicin does not block scission by the cuprous complexes of 3,4,7,8-tetramethyl-OP, 4-phenyl-OP, and OP but does perturb scission by the cuprous complex of 5-phenyl-OP. Organic ligands including intercalating agents and groove binders (e.g., daunomycin, di(amidinophenyl)indole (DAPI), actinomycin D, distamycin, 9-aminoacridine, mithramycin, and chromomycin A3), which bind to free DNA with high affinity, do not form stable ternary complexes with the open-complex. Gel retardation experiments demonstrate that they promote dissociation of the enzyme from the promoter. The greater sensitivity of enzymatic catalysis to inhibitor concentration relative to polymerase binding suggests that these ligands form metastable, catalytically inactive ternary complexes with RNA polymerase and the promoter.
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PMID:Interactions of transcription inhibitors with the Escherichia coli RNA polymerase-lacUV5 promoter open complex. 811 83

sigma 54 is a rare bacterial protein that substitutes for sigma 70 in the case of Escherichia coli genes transcribed by certain activators with enhancer protein-like properties. It contains a strongly acidic region of previously unknown function. Gel mobility-shift assays using sigma 54 deletion mutants show that this region is essential for sigma 54 to bind the core RNA polymerase and recruit it to the promoter. Multiple-point mutational analysis shows that the acidic amino acids and overlapping periodic hydrophobic amino acids are necessary for this binding. Related sequences are not found within the core binding determinant of sigma 70, which binds the same core RNA polymerase. This comparison suggests that the core RNA polymerase interacts differently with the two sigma factors, likely contributing to the critical differences in transcription mechanism in the two cases.
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PMID:RNA polymerase binding using a strongly acidic hydrophobic-repeat region of sigma 54. 813 58

The CynR protein, a member of the LysR family, positively regulates the Escherichia coli cyn operon and negatively autoregulates its own transcription. By S1 mapping analysis, the in vivo cynR transcription start site was located 63 bp upstream of the cynTSX operon transcription start site. Topologically, the cynR and cynTSX promoters overlap and direct transcription in opposite directions. The CynR translation initiation codon was identified by oligonucleotide-directed mutagenesis, and the CynR coding sequence was cloned under the control of a T7 phage promoter. The CynR protein was stably expressed at a high level with a T7 RNA polymerase-T7 phage promoter system. Purification by ion-exchange chromatography, affinity chromatography, and ammonium sulfate fractionation yielded pure CynR protein. Gel shift assays confirmed that CynR is a DNA-binding protein like the other members of the LysR family. The CynR regulatory protein binds specifically to a 136-bp DNA fragment encompassing both the cynR and the cynTSX promoters.
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PMID:Expression and purification of the cynR regulatory gene product: CynR is a DNA-binding protein. 825 86

Though widely recognized in higher eukaryotes, the regulation of Saccharomyces cerevisiae genes transcribed by RNA polymerase II by proteins that bind within the coding sequence remains largely speculative. We have shown for the LPD1 gene, encoding lipoamide dehydrogenase, that the coding sequence between +13 and +469 activated gene expression of an LPD1::lacZ fusion by up to sixfold in the presence of the upstream promoter. This downstream region, inserted upstream of a promoterless CYC1::lacZ fusion, activated gene expression in a carbon source-dependent manner by a factor of 15 to 111, independent of orientation. Deletion and mutational analysis identified two downstream activation sites (DAS1 and DAS2) and two downstream repressor sites (DRS1 and DRS2) that influence the rate of LPD1 transcription rather than mRNA degradation or translation. Activation from the DAS1 region (positions +137 to +191), encompassing a CDEI-like element, is twofold under derepressive conditions. Activation from DAS2 (+291 to +296), a CRE-like motif, is 12-fold for both repressed and derepressed states. DRS1, a pair of adjacent and opposing ABF1 sites (+288 to +313), is responsible for a 1.3- to 2-fold repression of transcription, depending on the carbon source. DRS1 requires the concerted action of DRS2 (a RAP1 motif at position +406) for repression of transcription only when the gene is induced. Gel mobility shift analysis and in vitro footprinting have shown that proteins bind in vitro to these downstream elements.
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PMID:Yeast intragenic transcriptional control: activation and repression sites within the coding region of the Saccharomyces cerevisiae LPD1 gene. 826 90


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