The two effects of rifampicin on the RNA polymerase reaction.

At 25°C rifampicin strongly stimulates the synthesis of the dinucleotide pppA-U catalyzed by the DNA-dependent RNA polymerase from $\text{Escherichia coli}$. If the antibiotic is added to the enzyme during the synthesis of RNA the stimulatory effect on the dinucleotide synthesis is distinctly retarded as is its inhibitory action on RNA synthesis. It is proposed that this lag period is due to a retardation of the binding of rifampicin to RNA polymerase which is required for its action. Because of this slower binding rifampicin — although an inhibitor of RNA chain elongation — mimics the action of an inhibitor of RNA chain initiation.     

The properties of ATP-analogs in initiation of RNA synthesis catalyzed by RNA polymerase from E coli.

Various base and sugar modified derivatives of ATP and UTP were used as substrate analogs for the steady state initiation reaction ATP+UTP=pppApU and the single step addition reaction ApC+ATP=ApCpA. These reactions were carried out by E. coli RNA polymerase on T7 DNA in the presence of rifampicin. The steady state kinetic parameters of the analogs, either as substrates or inhibitors, were determined. On the basis of the obtained results it is concluded that purine NTP s in initiation require anti-conformation about the glycosidic bonds as well as gauche-gauche conformation of the C(4')-C(5') bonds. The latter conformation is also a prerequisite for substrates in elongation, whereas strict anti-conformation of glycosidic bonds is not.     

On the mechanism of rifampicin inhibition of RNA synthesis.

The mechanism of rifampicin inhibition of Escherichia coli RNA polymerase was studied with a newly developed steady state assay for RNA chain initiation and by analysis of the products formed with several 5'-terminal nucleotides. The major effect of rifampicin was found to be a total block of the translocation step that would ordinarily follow formation of the first phosphodiester bond. These effects were incorporated into a steric model for rifampicin inhibition. Additional minor effects of the enzyme bound inhibitor were to increase slightly the lifetime of RNA polymerase on the lambdaPR' promoter and to increase by two the apparent Michaelis constants of the initiating triphosphates. The products formed by RNA polymerase in the presence of rifampicin belong nearly exclusively to the class pppPupN. No evidence for the accumulation of such molecules was obtained in vivo.     

RNA polymerase: interaction of RNA and rifampicin with the subassembly alpha 2 beta

We studied the inhibition of tryptic digestion of the subassembly alpha 2 beta of Escherichia coli DNA-dependent RNA polymerase to investigate its interaction with RNA and rifampicin. Both agents decreased distinctly the cleavage of subunit beta in the subassembly as well as the degradation of the transiently formed polypeptides (Mr greater than 80000). Short RNAs with a chain length of approximately 35 nucleotides were most protective at a concentration of 1 mg/ml while long RNAs were less effective at the same concentration. DNA did not exert any observable protective effects. The association of RNA with alpha 2 beta was shown by chromatography on phosphocellulose, which separates alpha 2 beta bound to RNA from free alpha 2 beta. The association of alpha 2 beta with RNA was inhibited by rifampicin.     

A steady state assay for the RNA polymerase initiation reaction.

A new assay yielding mechanistic information on the initiation reaction of Escherichia coli RNA polymerase has been developed. It was found to be useful in characterizing the promoters of bacteriophage DNA templates. The binding of the first two triphosphates in an RNA sequence was determined to be equilibrium ordered with ATP binding first followed by UTP on the lambda promoters PL. and PR. The products resulting from phosphodiester bond formation, pppApU and PPi, dissociated rapidly in the absence of the other triphosphates required for RNA synthesis. The resulting steady state conversion of ATP and UTP into pppApU was the basis for the new assay. The rate-limiting step in the initiation reaction was not precisely determined, but it was argued not to be entirely the release of product. The Zn2+ chelator, 1,10-phenanthroline, was partially characterized and found to be an uncompetitive inhibitor of ATP in the reaction (Ki = 100 micrometer). The unique advantage of this steady state assay is that several steps in the RNA initiation process are amplified kinetically and thus can be examined separately with techniques applicable to any other two-substrate, two-product enzyme reaction.     

The effect of low substrate concentrations on the extent of productive RNA chain initiation from T7 promoters A1 and A2 by Escherichia coli RNA polymerase

The extent of productive RNA chain initiation in vitro by Escherichia coli RNA polymerase holoenzyme from the bacteriophage T7 A1 and A2 promoters on purified T7 DNA templates has been investigated at very low concentrations of the ribonucleoside triphosphate substrates. As the concentration of ribonucleoside triphosphates in the reaction is decreased from 10 to 1 micro M, the extent of productive RNA chain initiation at these promoter sites drops precipitously at about 3 micro M. At 1 micro M substrate concentration, productive chain initiation from the A1 promoter does not occur even after extended incubation although the dinucleoside tetraphosphate pppApU is produced at a significant rate under these conditions. The reason for the inability of RNA polymerase to carry out productive RNA chain initiation at 1 micro M substrate concentration is not yet understood. The phenomenon is not due to substrate consumption, enzyme inactivation, or a requirement for a nucleoside triphosphatase activity in the reaction. The possibility is raised that there are additional nucleoside triphosphate binding sites on E. coli RNA polymerase which play some role in the process of productive RNA chain initiation.     

Micro-analysis of pure deoxyribonucleic acid-dependent ribonucleic acid polymerase from Escherichia coli. Action of heparin and rifampicin on structure and function

By using micro disc electrophoresis and micro-diffusion techniques, the interaction of pure DNA-dependent RNA polymerase (EC 2.7.7.6) from Escherichia coli with the template, the substrates and the inhibitors heparin and rifampicin was investigated. The following findings were obtained: (1) heparin converts the 24S and 18S particles of the polymerase into the 13S form; (2) heparin inhibits RNA synthesis by dissociating the enzyme-template complex; (3) rifampicin does not affect the attachment of heparin to the enzyme; (4) the substrates ATP and UTP are bound by enzyme loaded with rifampicin; (5) rifampicin is bound by an enzyme-template complex to the same extent as by an RNA-synthesizing enzyme-template complex. From this it is concluded that the mechanism of the inhibition of RNA synthesis by rifampicin is radically different from that by heparin. As a working hypothesis to explain the inhibitory mechanism of rifampicin, it is assumed that it becomes very firmly attached to a position close to the synthesizing site and only blocks this when no synthesis is in progress.     

DNA-dependent RNA polymerase from an extremely thermophilic hydrogen-oxidizing bacterium Calderobacterium hydrogenophilum

Extremely thermophilic bacterium Calderobacterium hydrogenophilum contains DNA-dependent RNA polymerase with unusual properties. Purified enzyme is thermoresistant (40 min at 100 degrees C) and exhibits similar subunit composition as eubacterial RNA polymerases (e.g. Escherichia coli). However, the enzyme is not susceptible to antibiotics which inhibit eubacterial RNA polymerases (rifampicin and streptolydigin). The activity of the enzyme is inhibited by actinomycin D, daunomycin and heparin.     

Inhibition of RNA synthesis in vitro by 9-aminoacridine carboxamide antitumor agents. Effects on overall RNA synthesis and synthesis of the initiating dinucleotide.

A series of 9-aminoacridine carboxamide derivatives of systematically varied structure was assayed in an RNA synthesis in vitro system. Escherichia coli DNA-dependent RNA polymerase and DNA derived from phage T7 or calf thymus were used to measure the effect of the drugs on overall RNA and the initiating dinucleotide (pppApU) syntheses. By means of multiple linear regression analysis it was shown that the inhibition of these reactions depends both on the drug equilibrium binding constant and kinetic parameters of dissociation of drug-DNA complexes.     

Interaction of RNA polymerase mutations in haploid and merodiploid cells of Escherichia coli K-12

Summary Combination in the same chromosome of tsX mutation which affects the attachment of RNA polymerase to DNA template with either of two rifampicin resistant mutations (rif-r-1 or rif-r-5) is lethal. However tsX forms viable combination with other rifampicin resistant mutation—rif-r-76. Moreover a partial restoration of rifampicin binding capacity takes place: RNA polymerase from double tsX rif-r-76 mutant binds rifampicin better than the enzyme from tsX ⁺ rif-r-76 cells. No mutual influence of rifampicin resistant and streptolydigin resistant mutations was found.Heterozygous merodiploids (rif-r/rif-s and stl-r/stl-s) demonstrate phenotypic dominance of sensitivity to each of the drugs no matter whether resistant allele is localized in chromosome or in episome. However certain chromosomal mutations which themselves have no apparent effect on RNA polymerase may cause dominance of rif-r allele.About a half of total cellular RNA polymerase in crude extracts of rif-r/rif-s and stl-r/stl-s heterogenotes was found to be drug-resistant, though rif-s allele is dominant phenotypically.The development of T2 phage is completely inhibited by rifampicin in haploid rif-s cells and is only slightly affected in rif-r mutant. A partial resistance of phage development to rifampicin was observed in rif-r/rif-s heterogenotes which confirms that both rif-s and rif-r enzymes are simultaneously present in such cells.Sensitive and resistant RNA polymerase function independently when mixture of the two enzymes was incubated with the excess of DNA template. However a competition between the two enzymes for the DNA was observed if the limiting amount of the template is available. The result of this competition to major extent depends on which of the enzymes was added first. It is supposed that in certain conditions normal RNA polymerase may act as a repressor of the mutant enzyme: drug-sensitive RNA polymerase may bind to the template in the presence of the drug and thus prevent the function of drug-resistant enzyme. This hypothesis explains phenotypic dominance of sensitive alleles to resistant alleles which leads to inability of heterogenote cells to multiply in the presence of corresponding drugs.