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)

Urea, at concentrations which do not interfere with bacterial growth, specifically inhibits the expression of catabolite sensitive operons. To search for the target and the mechanism of urea action we measured lactose (lac) and tryptophanase (tna) specific mRNA synthesis in vivo and in vitro. We show that urea acts by two different mechanisms at these two catabolite sensitive operons, resembling the manner in which catabolite repression regulates lac and tna. At the lac promoter, urea abolishes transcription initiation or blocks an early step in mRNA elongation without interfering with the binding of RNA polymerase and catabolite gene activator protein (CAP). At the tna promoter, urea does not abolish transcription initiation but could interfere with tnaC translation.
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PMID:Two different mechanisms for urea action at the LAC and TNA operons in Escherichia coli. 216 52

By use of techniques described recently for lac permease [Roepe, P.D., & Kaback, H.R. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6087], the melibiose permease from Escherichia coli, another polytopic integral plasma membrane protein, has been purified in a metastable soluble form after overexpression of the melB gene via the T7 RNA polymerase system. As demonstrated with lac permease, soluble melibiose permease is dissociated from the membrane with 5.0 M urea and appears to remain soluble in phosphate buffer at neutral pH after removal of urea by dialysis, although the protein aggregates in a time- and concentration-dependent fashion. Moreover, soluble melibiose permease behaves as a monomer during purification by size exclusion chromatography in the presence of urea. Circular dichroism of purified soluble melibiose permease reveals that the protein is highly helical in potassium phosphate buffer and that secondary structure is disrupted in 5.0 M urea. Finally, purified melibiose permease can be reconstituted into proteoliposomes, and the preparations catalyze membrane potential driven H+/melibiose or Na+/methyl 1-thio-beta,D-galactopyranoside symport. The results provide further support for the notion that hydrophobic transmembrane proteins may be able to assume a nondenatured conformation in aqueous solution and extend the implication that the approach described may represent a general method for rapid isolation and reconstitution of this class of membrane proteins.
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PMID:Isolation and functional reconstitution of soluble melibiose permease from Escherichia coli. 218 31

HO-221, N-[4-(5-Bromo-2-pyrimidinyloxy)-3-chlorophenyl]-N'-(2-nitrobenzoyl ) urea is a novel benzoylphenylurea derivative. We previously reported HO-221 showed significant antitumor activities against various experimental tumor models, and was especially effective against the solid tumor. In this report we studied the mechanism of action of the compound. The inhibitory activity of HO-221 and 6 kinds of antitumor agents on DNA polymerase alpha was examined in vitro. HO-221 inhibited DNA polymerase alpha activity strongly. From the comparison with IC50 values of individual agents, the inhibitory activity of HO-221 was almost equivalent to aphidicolin and ara-CTP. By double reciprocal plot analysis, the inhibition of HO-221 was found to be non-competitive with the dCTP unlike that of aphidicolin and ara-CTP. Furthermore, HO-221 showed almost no effect on RNA polymerase activity and the protein synthesis. The effect of HO-221 on cell cycle progression of HL-60 cells was examined by flow cytometry analysis. The compound accumulated cells at S phase at a low concentration. The compound showed accumulation of cells in G1, G1-S and G2 + M phases. At higher concentrations, HO-221 increased the G1 phase of tumor cells, stopping the cell cycle progression. Therefore, G1 and S phase accumulation by HO-221 was considered to be correlated with the inhibition of DNA polymerase alpha dependent DNA synthesis. These results suggest that HO-221 is a novel antitumor agent with different mechanism of action from the known antitumor agents.
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PMID:[Mechanism of antitumor effect of a benzoylphenylurea derivative, HO-221]. 226 Aug 70

Two urea-free agarose gel protocols that resolve the six individual strands of bacteriophage phi 6 dsRNA were developed and used to analyze phage RNA synthesis in vivo and in vitro. Citrate gels separate strands of the large and medium chromosomes while Tris-borate-EDTA (TBE) gels resolve the medium and small dsRNA segments. Minus strands migrate faster than plus strands on citrate gels but are retarded on TBE gels. A study of electrophoretic conditions showed that pH affects strand resolution on citrate gels, and that voltage gradient, agarose concentration, and ethidium bromide significantly alter strand migration on TBE gels. Analysis of native phi 6 RNA synthesized in vivo and in vitro showed that the large and medium message RNAs comigrate with the corresponding plus strands of denatured virion dsRNA. The small messenger RNA is exceptional. Native small mRNA was detected as three isoconformers in vivo and in vitro. The isoconformers were converted by heat denaturation to a single RNA species that comigrates with the virion s+ strand. Minus strands labeled in vivo were detected only after heat denaturation. Minus strand synthesis was detected also in heat-denatured samples from in vitro phi 6 nucleocapsid RNA polymerase reactions at pH values suboptimal for transcription.
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PMID:Detection of bacteriophage phi 6 minus-strand RNA and novel mRNA isoconformers synthesized in vivo and in vitro, by strand-separating agarose gels. 235 55

Since previous studies have suggested that the mammalian protamine mRNAs are translated poorly in cell-free systems, we directly measured the efficiency of translation of mouse protamine 1 mRNA. We found that mouse testis poly(A)+ mRNA stimulates the synthesis in the wheat germ and reticulocyte cell-free systems of three prominant translation products which can be resolved by electrophoresis through acid urea polyacrylamide gels containing 8 M urea. These translation products have been identified as testis-specific protein, protamine 1, and the precursor to protamine 2 by several criteria, including labeling with amino acids, [35S]cysteine, and [3H]leucine, which are known to be specific to some of these proteins from the nucleotide sequences of recombinant DNAs. Surprisingly, the mobility of the testis-specific protein translation product is slightly reduced and the mobility of both protamine translation products is drastically reduced unless the extracts of cell-free translations are coelectrophoresed with the appropriate carrier. The fraction of [35S]cysteine- labeled protamine 1 translation product was compared with the fraction of testis poly(A)+ mRNA as protamine 1 mRNA which we measured in dot blots with the use of an SP6 RNA polymerase transcript for protamine 1. The results demonstrate that protamine 1 mRNA is translated only slightly less efficiently than the average testis poly(A)+ mRNA.
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PMID:Translation of mouse testis poly(A)+ mRNAs for testis-specific protein, protamine 1, and the precursor for protamine 2. 244 49

Pleiotropic mutants of Alcaligenes eutrophus with the phenotype Hno- have been characterized previously. They are deficient in several diverse metabolic activities, including hydrogen oxidation, nitrate and urea assimilation, denitrification, and various substrate transport systems. Phenotypically similar mutants were identified among hydrogenase-deficient strains of Pseudomonas facilis. The Tn5-labeled hno gene was cloned from a genomic DNA library of A. eutrophus and used to identify the corresponding unimpaired wild-type DNA sequence. The recombinant plasmid pCH148 contained an insert of 12.3 kilobase pairs and was shown to restore the Hno+ phenotype to mutants of A. eutrophus and P. facilis. A cosmid isolated from a DNA library of P. facilis also exhibited intergeneric Hno-complementing activity. The cloned hno loci from both organisms showed DNA homology by Southern blot hybridization. A subclone of pCH148 which contained a 6.5-kilobase-pair insert was constructed. The resulting hybrid, pCH170, not only was able to complement Hno- mutants but also relieved glutamine auxotrophy in NtrA- mutants of enteric bacteria. This suggests that the hno gene product from A. eutrophus is functionally similar to the NtrA protein, which has been identified as a novel sigma factor (sigma 54) of RNA polymerase.
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PMID:An rpoN-like gene of Alcaligenes eutrophus and Pseudomonas facilis controls expression of diverse metabolic pathways, including hydrogen oxidation. 253 72

Only three forms of Kunjin virus-specified RNA were isolated from cytoplasm early after the latent period (about 15 hr) viz., 44 S genomic-sized single-stranded RNA, 20 S double-stranded "replicative form" (RF), and 20-28 S partially ribonuclease-resistant (about 70%) "replicative intermediate" (RI). The RF and RI were resolved by electrophoresis in aqueous-agarose gel only following LiCl fractionation. The RI did not enter urea-polyacrylamide gels. After denaturation of untreated or RNase-treated RI and RF, only 44 S RNA was present in electropherograms. RNA polymerase activity at 8 hr postinfection was detected by in vitro assays of cytoplasmic extracts and reached a maximum at 24 hr, the only major labeled product being RF; a trace amount of free 44 S RNA was also produced. These results, and the kinetics of incorporation of [3H]uridine into RI, RF, and 44 S RNA in pulse and pulse-chase experiments, formed the basis of a model in which flavivirus RF functions as a recycling template for semiconservative and (mainly) asymmetric replication, on which only one nascent strand is synthesized per cycle.
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PMID:Replication strategy of Kunjin virus: evidence for recycling role of replicative form RNA as template in semiconservative and asymmetric replication. 257 39

Lac permease, a polytopic membrane protein from Escherichia coli, has been purified in soluble form by overexpressing the lacY gene by means of the T7 RNA polymerase system. Soluble permease is dissociated from membranes with urea or other chaotropes and appears after the membrane is saturated with newly synthesized permease. Remarkably, this form of the permease appears to remain soluble in phosphate buffer at neutral pH after removal of urea, although it aggregates in a time- and concentration-dependent manner. Importantly, soluble permease behaves as a monomer during size-exclusion chromatography with or without urea, contains less than 3 mol of organic phosphate per mol of protein, and is largely helical. Soluble permease binds p-nitrophenyl alpha-D-galactopyranoside approximately 40% as well as permease in the native environment of the membrane and can be reconstituted into phospholipid vesicles that catalyze lactose counterflow or active transport in response to a membrane potential (interior negative). The results suggest that lac permease can assume a nondenatured conformation in aqueous solution.
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PMID:Characterization and functional reconstitution of a soluble form of the hydrophobic membrane protein lac permease from Escherichia coli. 266 55

We have cloned and expressed in Escherichia coli the gene encoding the trimeric fiber protein of human adenovirus type 2. A gene expression system based on bacteriophage T7 RNA polymerase was used. Optimal gene expression was obtained with 1-h induction, at a temperature of 30 degrees C. The synthesized protein constituted about 1% of total host-cell protein. During induction, the growth of bacteria carrying the plasmid containing the fiber gene, was retarded compared with that of bacteria carrying the plasmid without the fiber gene. This toxic effect of fiber protein on bacterial hosts could be diminished by addition of glucose to the medium and by maintaining the pH above 7, thus improving the yield of recombinant fiber protein. The fiber protein produced in E. coli is stable during the course of induction. It is insoluble in buffers at physiological pH, in various salt solutions, and in the presence of nonionic detergents. It can be solubilized in 1% sodium dodecyl sulfate or in urea solutions above 2 M. There are indications that recombinant fiber trimerizes spontaneously, since after the removal of urea by dialysis at pH 8, recombinant fibers runs similarly to native trimeric fiber, on nondenaturing polyacrylamide gels. This trimer has, however, a less compact structure than native Ad2 fiber, since during gel filtration recombinant protein is excluded before native protein. It is also more sensitive to chymotrypsin digestion than native fiber.
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PMID:Synthesis of human adenovirus type 2 fiber protein in Escherichia coli cells. 268 Jul 70

The thr operon of Escherichia coli is regulated by an attenuation mechanism in which regulated transcription termination occurs in response to the levels of charged tRNAthr and tRNAile. Transcription of the thr operon regulatory region in vitro produces a 162-base transcript that is terminated efficiently at the attenuator. The attenuator sequence is similar to other rho-independent terminators. It contains a G + C region of dyad symmetry followed by a run of 9 A + T residues. We have characterized in detail the sequence requirements for efficient transcription termination in vitro. Using a set of point mutations in the G + C region of dyad symmetry of the thr attenuator, we have characterized the effects of these mutations on the efficiency of transcription termination. The efficiency was reduced in all of the mutants analyzed with the greatest effect being an approximate 20% decrease in termination. In some instances the electrophoretic mobilities of the terminated transcripts on 8% polyacrylamide, 8 M urea gels were shifted substantially relative to the wild type-terminated transcript, but the sites of transcription termination were altered by only a few base pairs. We also constructed a set of deletions removing consecutive thymidines which follow the G + C-rich region of dyad symmetry. Removal of 1 or 3 of the 9 thymidine residues had no effect on termination efficiency in vitro or in vivo. Removal of four to six thymidines caused a linear decrease in the efficiency of termination. When only one or two thymidines were present in the template, termination was completely abolished. These results indicate that both the integrity of the RNA stem and the length of the consecutive thymidine residues are important signals recognized by RNA polymerase during transcription of the thr operon regulatory region.
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PMID:Contributions of RNA secondary structure and length of the thymidine tract to transcription termination at the thr operon attenuator. 296 47


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