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 in vitro synthesis of elongation factor (EF)-Tu (tufB), the beta beta' subunits of RNA polymerase, ribosomal proteins L10 and L12 directed by DNA from the transducing phage lambda rifd 18, EF-Tu (tufA), EF-G, and the alpha subunit of RNA polymerase directed by DNA from the transducing phage lambda fus3 has been investigated in a crude and a partially defined protein-synthesizing system. Proteins L10 and L12 are synthesized in the partially defined system almost as well as in the crude system. However, the synthesis of EF-Tu, EF-G, and the alpha and beta beta' subunits of RNA polymerase is far less efficient in the partially defined system. An active fraction that stimulates the synthesis of these latter proteins has been obtained by fractionation of a high-speed supernatant on DEAE-cellulose. Because previous studies showed that this fraction (1 M DEAE salt eluate) contains a protein, called L factor, that stimulates beta-galactosidase synthesis in vitro, L factor was tested for activity. Although L factor stimulates the synthesis of the beta beta' subunits, it has little or no effect on the in vitro synthesis of the other products studied. In the present experiments, the ratio of L12/L10 and of EF-Tu (tufA)/EF-G formed is 4-6. These values are consistent with in vivo results.
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PMID:DNA-directed in vitro synthesis of proteins involved in bacterial transcription and translation. 16 May 61

The rate of differential synthesis of beta-galactosidase (alphalac) was measured in maximally induced cultures of Escherichia coli B/r with 0.01 M-inducer and 0.01 M-cyclic AMP. The value of alphalac decreases with growth rate (60% between 0.67 and 2.1 doublings/h) and after a nutritional shift-up. This decrease is presumed to reflect a decrease in the intracellular concentration of free active RNA polymerase after a shift-up, which implies that the increase in ribosome synthesis after a shift-up is due to an active induction of the ribosomal components.
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PMID:Metabolic regulation of beta-galactosidase synthesis in Escherichia coli. A test for constitutive ribosome synthesis. 17 97

Three temperature-sensitive mutant strains for RNA polymerase beta or beta' subunits (carrying mutations tsx, A2R7 and R120) were used in order to investigate the dependence of the induced lac expression on stimulation by cyclic AMP after the shift to non-permissive temperature. High temperature lowered the rate of beta-galactosidase synthesis. However, the low rate of synthesis could be strongly increased by cyclic AMP (30, 2.4 and 5.7-fold increases for tsX, A2R7 and R120 mutants, respectively). At the permissive temperature stimulation by cyclic AMP was less than 1.4-fold (minimal medium supplemented with glycerol). The results suggest that the maximal expression of the lac operon is saturated, that is, a hypothetical increase in RNA polymerase or cAMP-CRP concentration in the cell with not enhance the expression. The concept of saturation explains why it was possible to increase the beta-galactosidase synthesis in conditions of limited promoter binding activity of RNA polymerase through increase in concentration of cyclic AMP-CRP complex in the cell (addition of cyclic AMP) to the values higher than that observed on glycerol.
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PMID:Expression of the lac operon in RNA polymerase mutants of Escherichia coli K12. 22 41

The expression of the genes coding for the beta and beta' subunits of RNA polymerase, ribosomal RNA, ribosomal proteins, and beta-galactosidase was investigated in strains carrying conditionally lethal mutations affecting either RNA polymerase core assembly or RNA polymerase enzyme activity. The mutant strain XH56 produces a temperature-sensitive beta' subunit and at 42 degrees C is defective in RNA chain initiation; consequently, little or no transcription occurs at the restrictive temperature. A partial restriction, produced by shifting the strain to 39 degrees C, resulted in a rapid fivefold increase in the transcription of the rpoB and C genes and in the synthesis of the beta- and beta'-subunit proteins for which they code. The RNA polymerase assembly-defective strains A2R7 and TS4 exhibited a 1.5- to 2-fold increase in the transcription of the rpoB and C genes and in the synthesis of beta- and beta-subunit proteins after prolonged restriction. These results demonstrate (i) that regulation of the synthesis of the beta- and beta-RNA polymerase subunits is under these conditions primarily transcriptional rather than translational, and (ii) that a stimulation of rpoB and C gene expression results from a restriction on RNA synthesis caused by either RNA polymerase inactivation or inhibition of its assembly. During restriction of the mutant strains, the transcription of the ribosome component genes exhibited patterns which were similar to transcription of the rpoB and C genes, supporting the evidence that genes coding for RNA polymerase are cotranscribed with ribosomal protein genes; transcription of the lacZ gene was observed to decrease concomitant with the stimulation of the rpoB and C genes.
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PMID:Expression of RNA polymerase and ribosome component genes in Escherichia coli mutants having conditionally defective RNA polymerases. 36 11

In an extract containing all the components for lac gene expression except washed ribosomes, lac mRNA formation was increased 4- to 6-fold by the addition of washed ribosomes. The formation of beta-galactosidase mRNA and enzyme showed very different dependency on added ribosomes. Enzyme was formed in proportion to the number of ribosomes added, whereas 10% of the standard level of ribosomes promoted full levels of transcription. Consistent with their action in vivo, chloramphenicol and erythromycin blocked the ribosome-dependent lac transcription. The same inhibition was seen with RNA pulse-labeled for 1 or 5 min, so that the effect was truly a blockage of formation rather than an increased hyperlability of nascent mRNA. The effect was specified for some RNA species, as it is in vivo: phage lambda N gene transcription was increased rather than inhibited in the presence of chloramphenicol. Chloramphenicol did not stop lac transcription as a result of its blockage of formation of the regulatory nucleotide tetraphosphate (ppGpp), because addition of the nucleotide did not restore mRNA formation in chloramphenicol-treated extracts. Rather, the data are consistent with the ideas that one or a few ribosomes moving closely behind RNA polymerase can prevent its arrest and that, when ribosome movement is blocked by chloramphenicol, the RNA polymerase is exposed to factors that provoke premature RNA chain termination.
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PMID:Coupling of lac mRNA transcription to translation in Escherichia coli cell extracts. 41 5

The phage DNA-directed synthesis of beta-galactosidase has been examined in a system containing the following purified Escherichia coli factors: RNA polymerase; cyclic AMP receptor protein; N10-formyltetrahydrofolate Met-tRNAf transformylase; initiation factors 1, 2, and 3; elongation factors Tu, Ts, and G; release factors 1 and 2; 20 aminoacyl-tRNA synthetases; L factor (Kung, H. F., Spears, C., and Weissbach, H. (1974) J. Biol. Chem. 250, 1556-1562); and Lalpha (Kung, H.-F., Spears, C., and Weissbach, H. (1976) Fed. proc. 35, 1537). Under these conditions, beta-galactosidase synthesis occurs at less than 1% of the rate obtained with unfractionated extracts, which suggested that other required components were lacking. The difficulty in obtaining large amounts of the purified aminoacyl-tRNA synthetases for these studies made it necessary to modify the system. It was possible to conserve many of the purified aminoacyl-tRNA synthetases since at least 13 of them could be replaced by an Ehrlich ascites extract. The ascites extract plus other E. coli purified factors was used as a basic system to search for additional components required for beta-galactosidase synthesis. The present report describes the purification from E. coli extracts of three fractions, called Lbeta, Lgamma, and Ldelta, that are needed to restore enzyme synthesis.
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PMID:DNA-directed in vitro synthesis of beta-galactosidase. Studies with purified factors. 56 Oct 72

Four mutants of Escherichia coli KL16 resistant to the antibiotic Thiolutin have been isolated. This drug was earlier reported to be an inhibitor of RNA chain elongation. The first mutant, TLrI, is resistant only in rich or partially rich media: it can, however, grow in minimal medium containing the drug with a very long doubling time. The other mutants TLrII, TLrIIIa and TLrIIIb are resistant in rich as well as minimal media. beta-galactosidase could not be induced in TLrI and TLrII in the presence of thiolutin whereas the enzyme is constitutively synthesised in TLrIIIa and TLrIIIb irrespective of the drug. The mutants do not support the development of phage T4 in presence of the drug, if the drug is added along with the phage, but "escape" the inhibition if phage development is allowed to proceed for some time before the addition of the drug. The time of this escape is characteristic of the mutant. Even in a sensitive strain, T7 growth escapes inhibition very soon after infection, around the time the phage-specific RNA polymerase is synthesized. In the parent strain the kinetics of inhibition of beta-galactosidase induction resembles more the inhibition caused by rifampicin than by streptolydigin. It is proposed that thiolutin could be an inhibitor of RNA chain initiation and resistance might be due to mutation in the subunit(s)/factor(s) involved in initiation.
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PMID:Thiolutin resistant mutants of Escherichia coli are they RNA chain initiation mutants? 77 14

A strain of Escherichia coli has been isolated from a E. coli HfrH after a treatment with nitrosoguanidine for a partial resistance to thymineless death. This strain shows, in addition, a global increase of the RNA content amounting to 50 per cent. The half-life of the unstable RNA as well as of the messenger RNA of the beta-galactosidase is increased to a considerable degree. The RNA polymerase of this strain is modified with respect to its resistance to rifampicin and its transcription efficiency in vitro of various DNA templates. Our working hypothesis is that the primary alteration of this strain lies at the RNA polymerase level with a consequent increase of the synthetic rate of the ribosomal RNA. The increase of the messenger RNA half-life may be an indirect consequence of this event via a saturation of the RNAase by the excess of ribosomal RNAs not associated to their proteins.
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PMID:[Escherichia coli K12 mutant with increased RNA content and messenger RNA stability]. 78 59

E. coli fMet-tRNAfMEet and E. coli RNA plymerase (RNA nucleotidyltransferase; EC 2.7.7.6; nucleoside-triphosphate:RNA nucleotidyltransferase) form a 1:1 complex with an apparent association constant of 9.0 X 10(6)M-1 at 37 degrees. The affinity of polymerase to tRNA depends on the tRNA as well as the formyl methionine moiety. Core polymerase has a greatly reduced affinity for initiator tRNA. Optimal binding conditions are similar to those that are also optimal for binding initiator tRNA to ribosomes. Binding of initiator tRNA to polymerase stimulates the transcription of lambda plac DNA, as determined in a crude cell-free system for beta-galactosidase (EC 3.2.1.23; beta-D-galactoside galactohydrolase) synthesis as well as in a highly purified transcription system.
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PMID:Specific binding of formylated initiator-tRNA to Escherichia coli RNA polymerase. 78 83

We describe a procedure that allows cysteine and methionine content to be determined on microgram amounts of partially purified protein. The only requirements are that the protein can be obtained as a pure band after electrophoresis on a polyacrylamide gel and that some data on amino acid content be available. This method involves double labeling by growing bacterial cells with [3H]leucine and [35S]SO4 and determining the ratio of these radioisotopes incorporated into the ribonucleic acid polymerase subunits. The relative specific activities of [3H]leucine and [35S]cysteine and methionine are determined from the ratio of these isotopes incorporated into beta-galactosidase, the leucine, cysteine, and methionine contents of which are known. We have used this procedure to determine the sulfur content of the subunits of Escherichia coli ribonucleic acid polymerase. These new data are necessary to quantitate the rates of synthesis of these subunits by in vivo labeling with [35S]SO4.
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PMID:Cysteine and methionine content of the Escherichia coli ribonucleic acid polymerase subunits. 78 43


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