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 fraction of the total ribonucleic acid (RNA) synthesis rate that is messenger RNA (mRNA) for ribosomal protein (r-protein) and ribosomal RNA (rRNA) has been estimated in valS(Ts) rel+ stringent and valS(Ts) relA1 relaxed strains of Escherichia coli during a partial inhibition of valyl-transfer RNA aminoacylation. The partial inhibition was accomplished by shifting the strains from the permissive growth temperature of 29.5 degrees C to the semipermissive temperature of 35.5 degrees C. The RNA synthesized at the elevated temperature was pulse labeled with [3H]uracil. The fraction of the total incorpoarted 3H radioactivity in r-protein mRNA or in rRNA was estimated by specific hybridization to the transducing phages gammaspc1, which carries about 15 r-protein genes and lambdailv5, which carries an rRNA transcription unit. The results clearly demonstrate that the rel gene influences the fraction of the total RNA synthesis rate that is r protein mRNA and rRNA; in the rel+ strain they are significantly increased relative to control cultures. This indicates that the expression of the genes coding for the RNA and protein component of the ribosome are most likely regulated at the level of transcription. Furthermore, it appears that the distribution of functioning RNA polymerase between rRNA genes, r-protein genes, and other types of genes is influenced by the rel gene control system; presumably this influence is mediated through the unusual nucleotide guanosine tetraphosphate.
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PMID:Influence of the stringent control system on the transcription of ribosomal ribonucleic acid and ribosomal protein genes in Escherichia coli. 32 Jan 85

The effect of low chloramphenicol concentrations on the biosynthesis of RNA, ribosomal proteins and RNA polymerase in E. coli CP 78 cells was studied. When protein synthesis was decreased by 50--70%, 14C-uracil incorporation in DNA increased twice, the rRNA synthesis being stimulated preferentially. In the presence of antibiotic the RNA/DNA ratio increased from 5,7 to 13,3. The differential rate of r-protein synthesis increased simultaneously with the stimulation of rRNA synthesis, so that alphar rises from 0,083 (without antibiotic) to 0,122 and 0,161 at 5 and 10 microgram/ml of chloramphenicol, respectively. The inhibition of protein synthesis by chloramphenicol is accompanied also by the increase of differential rate of synthesis of beta and beta' subunits of RNA polymerase. In the presence of 5 and 10 microgram/ml of chloramphenicol, alphap increased from 0,90% to 1,44 and 1,57%, respectively. It is assumed that the genes for beta and beta' subunits of RNA polymerase as the ribosomal genes are negatively controlled by guanosine tetraphosphate which intracellular concentration decreased in the presence of chloramphenicol. The known data on the influence of streptolydigin and rifampicin on the RNA polymerase biosynthesis are discussed in view of proposed hypothesis.
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PMID:[The influence of chloramphenicol on synthesis of ribosomes and beta and beta' subunits of RNA polymerase]. 33 37

Synthetic DNA templates were transcribed by Escherichia coli RNA polymerase using nucleoside 5'-[gamma-S]triphosphates as one of the nucleotide substrates. Substitution of the thiol analogues for the normal nucleotides had no effect on the rate of RNA synthesis. RNA synthesized with either adenosine 5'-[gamma-S]triphosphate or guanosine 5'-[gamma-S]triphosphate was isolated with high efficiency on mercury-agarose columns prepared by activation with low concentrations of cyanogen bromide. Sulfur was shown to be incorporated at the 5' end of RNA by identification of the tetraphosphate HSpppA32p liberated after alkaline hydrolysis of HS(A-32pU)n (alternating copolymer synthesized by the action of E. coli RNA polymerase on d(A-T)n-d(A-T)n with adenosine 5'-[gamma-S]triphosphate and uridine 5'-[alpha-32P]triphosphate as substrates). Transcripts elongated but not initiated with these thiol analogues did not bind to the affinity column. This technique provides an extremely sensitive assay for RNA synthesis initiation in vitro, since initiated transcripts containing radiolabel throught the entire transcript can be isolated.
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PMID:Incorporation of purine nucleoside 5'-[gamma-S]triphosphates as affinity probes for initiation of RNA synthesis in vitro. 33 43

Expression of the histidine (his) operon in Salmonella typhimurium was found to be positively correlated with the intracellular level of guanosine tetraphosphate (ppGpp). Limitation for amino acids other than histidine elicited a histidine-independent metabolic regulation of the operon. In bacteria grown at decreased growth rates, his operon expression was metabolically regulated up to a point, after which further decreases in growth rate no longer resulted in further enhancement of operon expression. Studies using strains carrying various regulatory and deletion mutations indicated that metabolic regulation is achieved predominantly by increased RNA chain initiations at the primary (P1) and internal (P2) promoters. Metabolic regulation ordinarly did not involve changes in RNA chain terminations at the attenuator site of the his operon. A model is proposed that involves ppGpp-induced changes in RNA polymerase initiation specificity at particular promoters. A second, special form of metabolic regulation may operate which also is histidine independent, but does involve relief of attenuation.
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PMID:Promoter- and attenuator-related metabolic regulation of the Salmonella typhimurium histidine operon. 34 9

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 inhibition of RNA polymerase with ATP and UTP analogues modified in the phosphate and ribose moieties has been investigated. 1. Modification of the terminal phosphate with a loss of the negative charge [adenosine 5'-(3-O-methyl)triphosphate, Ki = 1.75 mM] substantially weakens the binding ability of these analogues to the enzyme whereas modification with retention of the charge is not so detrimental [adenosine tetraphosphate, Ki = 0.17 mM]. 2. 2'-Modified analogues are only weak competitive inhibitors [2'-amino-2'-deoxyadenosine 5'-triphosphate, Ki = 2.3 mM] of their corresponding substrates [ATP, Km = 0.07 mM] whereas 3'-modified analogues are extremely potent in their inhibition [3'-amino-3'-deoxyadenosine 5'-triphosphate, Ki = 2.3 muM]. 3. A difference was observed in the inhibition of the elongation step of RNA polymerase by ATP and UTP analogues. Thus ATP analogues showed a strong binding to the CT form of the poly[d(A-T)] ternary complex and only a weak binding to the CA form. UTP analogues, on the other hand, showed a similar binding to both forms of the complex.
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PMID:Interaction of substrate analogues with Escherichia coli DNA-dependent RNA polymerase. 79 50

We have previously shown that the synthesis of ribosomal proteins (r proteins) in E. coli cells is under stringent control (Dennis and Nomura, 1974). Since guanosine tetraphosphate (ppGpp) has been implicated in stringent control, we examined the effects of ppGpp on the in vitro synthesis of r proteins directed by DNA from transducing phage lambdafus3 and lambdarifd18. lambdafus3 carries genes for protein elongation factors EF-Tu and EF-G, and RNA polymerase subunit alpha, in addition to genes for approximately 27 r proteins. lambdarifd18 carries genes for EF-Tu, RNA polymerase subunits beta and beta1, and a set of rRNAs, in addition to genes for approximately five r proteins. We have shown that low concentrations of ppGpp (0.2-0.3 mM) specifically inhibit DNA-dependent r protein synthesis in this system, and that this inhibition takes place directly, rather than as a consequence of the inhibition of rRNA synthesis by ppGpp. In addition, we have also shown that ppGpp inhibits the synthesis of EF-G, EF-Tu, and RNA polymerase subunit alpha, as well as rRNAs.
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PMID:DNA-dependent in vitro synthesis of fibosomal proteins, protein elongation factors, and RNA polymerase subunit alpha: inhibition by ppGpp. 99 Dec 74

A cell-free system derived from E. coli is described in which mature-sized 16S and 23S ribosomal RNAs (rRNA) are synthesized at a high relative rate, comprising 17-25% of the total transcription. The addition of guanosine tetraphosphate (ppGpp) to this system results in up to a 5-fold selective inhibition of rRNA accumulation. This effect is exerted at the level of synthesis rather than degradation. It is concluded that ppGpp, which is produced in large amounts by E. coli during amino-acid deprivation, could mediate the decrease in rRNA synthesis that accompanies such deprivation. The expression of other genes has also been investigated. No selective reduction of transfer RNA synthesis by ppGpp is observed. The trp and lac operons are found to be stimulated at the transcriptional level by the presence of this nucleotide. It is hypothesized that ppGpp interacts with the RNA polymerase in such a manner as to alter the affinity of the enzyme for promoters in an operon-specific fashion.
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PMID:Effects of guanosine tetraphosphate on cell-free synthesis of Escherichia coli ribosomal RNA and other gene products. 110 24

The kinetics of the RNA chain initiation reaction carried out by RNA polymerase bound to the initiator region of a DNA template have been analyzed. Initiation proceeds in a two-substrate reaction in which the initial binary complex (enzyme-DNA) is transformed into a ternary complex (enzyme-DNA-RNA) by formation of a dinucleoside tetraphosphate and release of inorganic pyrophosphate. In this reaction RNA polymerase serves as a reactant rather than acting catalytically. The concentration of the reacting binary complex decreases throughout the reaction; hence steady state approximations cannot be used. Kinetic equations for an ordered two-substrate reaction are derived. These are most useful for the special case of reaction in the presence of an inhibitor of initiation, such as rifampicin. Equations for the latter instance are solved exactly with recourse to the steady state approximation. It is found that measurements of the extent of the initiation reaction determined at different inhibitor and substrate concentrations can give information about the initiation reaction analogous to that obtained in standard steady state kinetic analysis. This theory is applied to the experimental study of the initiation reaction carried out by Escherichia coli RNA polymerase. It is found that the inhibitor rifampicin, which blocks the initiation reaciton, acts by binding to the same form of the binary complex as the nucleoside triphosphate substrate (ATP or GTP) which is incorporated into the 5' terminus of nascent RNA molecule. The binding of the 5'-terminal nucleoside triphosphate to the enzyme appears to be rate-limiting for the initiation reaction under standard assay conditions. Initiation appears to follow an ordered reaction mechanism; however, the order of addition of the two substrates is still uncertain.
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PMID:Kinetic analysis of ribonucleic acid chain initiation by Escherichia coli Ribonucleic acid polymerase bound to DNA. 110 16

A controversy has surrounded the questions of whether or not guanosine tetraphosphate (ppGpp) is a specific inhibitor of bacterial rRNA and tRNA synthesis, especially during normal exponential growth, and whether the RNA polymerase is the target of ppGpp action. To answer these questions, a pBR322-derived plasmid, pKT28, was constructed that carries the Escherichia coli relA gene encoding a ppGpp synthetase under control of the lacUV5 promoter. The plasmid was used to transform the ppGpp reporter strain VH271 in which expression of beta-galactosidase from an rrnB P1 promoter is inhibited by ppGpp. In the presence of high concentrations of lac inducer, bacteria of the transformed strain accumulate ppGpp with the result that synthesis of rRNA and beta-galactosidase is inhibited and growth ceases. At low concentrations of inducer, growth is only reduced and cells form small white colonies on X-gal indicator plates. After continued incubation, these colonies form blue sectors of faster growing mutant cells. Phage P1 transduction experiments showed that these mutants have mutations cotransducing with rpoB, the gene encoding the beta-subunit of RNA polymerase. One particular mutant strain, KT13, had acquired partial resistance to ppGpp inhibition of rRNA synthesis. The mutation in this strain was cloned by in vivo recombination into an rpoB plasmid. The presence of this plasmid conferred increased resistance to overproduction of ppGpp. These results suggest that ppGpp is a specific inhibitor of rRNA synthesis, even in the absence of amino acid starvation, and that RNA polymerase is involved as the target of ppGpp action.
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PMID:Toxic effects of high levels of ppGpp in Escherichia coli are relieved by rpoB mutations. 137 Aug 17


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