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.     

Sensitivity of DNA-repair-deficient strains of Escherichia coli to rifampicin killing

We have analyzed the role of RNA polymerase in DNA repair using the antibiotic rifampicin which binds specifically to the beta subunit of the enzyme. Several DNA-repair-deficient strains such as recA, uvr, and polA, and their isogenic parents were used for this study. All repair-deficient strains were found to be hypersensitive to rifampicin killing. Compared to the isogenic parent strains, recA strains are about 50 times more sensitive and the polA strain is about 100 times more sensitive to rifampicin killing. UvrA and uvrB strains are slightly more sensitive to rifampicin than the wild-type strains. The hypersensitivity of repair-deficient strains to rifampicin killing is totally abolished by the introduction of rifampicin-resistant mutations into these strains. We have examined the effect of rifampicin on RNA and protein synthesis in repair-deficient and -proficient strains. RNA and protein synthesis were found to be inhibited by rifampicin to the same extent among all the strains tested. The results also show that the resumption of DNA synthesis was significantly disrupted in DNA-repair-deficient strains following drug removal. Taken together these results suggest that RNA polymerase plays an essential role in DNA metabolism and such function may be replaced by polA and recA gene products and to a lesser extend by uvrA and uvrB gene products.     

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.     

Rifampicin Inhibition of Ribonucleic Acid and Protein Synthesis in Normal and Ethylenediaminetetraacetic Acid-Treated Escherichia coli

The kinetics of ribonucleic acid (RNA) and protein synthesis in rifampicin-inhibited normal and ethylenediaminetetraacetic acid (EDTA)-treated Escherichia coli was measured. Approximately 200-fold higher external concentrations of rifampicin were needed to produce a level of inhibition in normal cells comparable to that observed in EDTA-treated cells. The rates of RNA and protein synthesis in both kinds of cells decreased exponentially, after an initial lag phase, at all rifampicin concentrations tested. The lag phase was longer and the final exponential slope less for protein synthesis than for RNA synthesis at a given rifampicin concentration. Below certain rifampicin concentrations, both the lag phase and the subsequent exponential decrease in the rates of RNA and protein synthesis were found to be rifampicin concentration dependent. At greater concentrations only the time of the lag phase was decreased by higher rifampicin concentrations, whereas the slope of the exponential decrease in the rates of RNA and protein synthesis was unaffected. In all cases, the exponential decrease continued to at least a 99.8% inhibition of the original rate of synthesis. These in vivo results are consistent with the mode of rifampicin action determined from in vitro studies; rifampicin prevents initiations of RNA polymerase on deoxyribonucleic acid, but not its propagation, by binding the enzyme essentially irreversibly. The results also indicate the size distribution of messenger RNA molecules in E. coli under our conditions.     

Inadequate inhibition of host RNA polymerase restricts T7 bacteriophage growth on hosts overexpressing udk

Overexpression of udk, an Escherichia coli gene encoding a uridine/cytidine kinase, interferes with T7 bacteriophage growth. We show here that inhibition of T7 phage growth by udk overexpression can be overcome by inhibition of host RNA polymerase. Overexpression of gene 2, whose product inhibits host RNA polymerase, restores T7 phage growth on hosts overexpressing udk. In addition, rifampicin, an inhibitor of host RNA polymerase, restores the burst size of T7 phage on udk-overexpressing hosts to normal. In agreement with these findings, suppressor mutants that overcome the inhibition arising from udk overexpression gain the ability to grow on hosts that are resistant to inhibition of RNA polymerase by gene 2 protein, and suppressor mutants that overcome a lack of gene 2 protein gain the ability to grow on hosts that overexpress udk. Mutations that eliminate or weaken strong promoters for host RNA polymerase in T7 DNA, and mutations in T7 gene 3.5 that affect its interaction with T7 RNA polymerase, also reduce the interference with T7 growth by host RNA polymerase. We propose a general model for the requirement of host RNA polymerase inhibition.     

Selective inhibition of ribosomal RNA synthesis by rifampicin in E. coli cells]

Under partial inhibition of total RNA synthesis by rifampicin the formation of beta- and beta'-subunits of RNA polymerase is stimulated and the rRNA synthesis is selectively repressed. The differential rate of synthesis of the beta- and beta'-subunits increases from 1,15% up to 2,88% in the presence of 30 micrograms rifampicin per ml. Simultaneously the differential rate of rRNA synthesis decreases from 41% down to 10%. The degree of inhibition of rRNA synthesis by rifampicin depends on the cell growth rate.     

Effects of rifampicin, streptolydigin and actinomycin D on the replication of Col E1 plasmid DNA in Escherichia coli

We recently reported (Clewell et al., 1972) on an inhibitory effect of rifampicin on Col E1 plasmid replication. The present study represents a further characterization of this phenomenon as well as a study of the effects of two other known inhibitors of RNA synthesis, Streptolydigin and actinomycin D.During treatment of cells with chloramphenicol the colicinogenic factor E₁ (Col E1) continues to replicate for more than ton hours. During this time 4 to 5 S RNA is also synthesized. When varying concentrations of rifampicin were included during chloramphenicol treatment, inhibition of plasmid DNA synthesis correlated very closely with inhibition of cellular RNA synthesis. Similar experiments testing the effects of Streptolydigin and actinomycin D (during chloramphenicol treatment) showed that cellular RNA synthesis was at least 100 times more sensitive to these drugs than was plasmid DNA synthesis.When actively growing cells (i.e. cells not treated with chloramphenicol) were treated with a high concentration of rifampicin (250 μg/ml), chromosomal DNA synthesis continued to an extent representing about a 50% increase in DNA, while plasmid DNA synthesis appeared to stop abruptly.     

A resonance Raman study on the interaction of rifampicin with Escherichia coli RNA polymerase

The technique of resonance Raman spectroscopy has been used to investigate the interaction of the antibiotic rifampicin with Escherichia coli RNA polymerase. Spectra were analyzed by generating the first derivative of each recorded spectrum using the Savitsky-Golay algorithm. The only band that shifted significantly in the resonance Raman spectrum of rifampicin upon the formation of the drug-core polymerase complex was the amide III band. It underwent an 8 cm-1 shift from 1306 cm-1 in aqueous solution to 1314 cm-1. A comparable shift was observed for the rifampicin-holoenzyme complex. Thus, the interaction of the sigma subunit with the core polymerase does not significantly alter the manner in which rifampicin interacts with RNA polymerase. The nature of this shift has been analyzed further by recording the resonance Raman spectrum of rifampicin in a variety of solvents with different hydrogen-bonding solvents (benzene and carbon disulfide) the amide III band was observed at approximately 1220 cm-1; in dimethyl sulfoxide, a weak hydrogen-bond acceptor, 1274 cm-1; in water, a strong hydrogen-bonding solvent, 1306 cm-1; and finally, in triethylamine, a stronger hydrogen-bonding solvent than water, it was observed at 1314 cm-1. Thus, as the hydrogen-bonding ability of the solvent increased, the amide III band shifted to higher frequency. Based on these results, the rifampicin binding site in RNA polymerase provides a stronger hydrogen-bonding environment for the amidic proton of rifampicin than is encountered when rifampicin is free in aqueous solution.     

Isolation and characterization of a mitochondrial RNA polymerase from Drosophila melanogaster

A DNA-dependent RNA polymerase was solubilized from sucrose gradient isolated, DNase-treated mitochondria of Drosophila melanogaster. The isolated mitochondria were not detectably contaminated with nuclear DNA as shown by CsCl gradient centrifugation and polylysine Kieselguhr chromatography. The detergent-solubilized RNA polymerase was sensitive to rifampicin, resistant to alpha-amanitin, had an apparent molecular mass of about 60 kilodaltons, and displayed a tendency to aggregate, both in crude extracts or when purified. The mitochondrial RNA polymerase could be distinguished from nuclear RNA polymerases on the basis of size, salt optima, rifampicin sensitivity, and alpha-amanitin resistance.     

The influence of chloramphenicol on synthesis of ribosomes and beta and beta' subunits of RNA polymerase]

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.     

Mapping and sequencing of mutations in the Escherichia coli rpoB gene that lead to rifampicin resistance

Rifampicin is an antibiotic that inhibits the function of RNA polymerase in eubacteria. Mutations affecting the beta subunit of RNA polymerase can confer resistance to rifampicin. A large number of rifampicin-resistant (hereafter called Rifr) mutants have been isolated in Escherichia coli to probe the involvement of RNA polymerase in a variety of physiological processes. We have undertaken a comprehensive analysis of Rifr mutations to identify their structural and functional effects on RNA polymerase. Forty-two Rifr isolates with a variety of phenotypes were mapped to defined intervals within the rpoB gene using a set of deletions of the rpoB gene. The mutations were sequenced. Seventeen mutational alterations affecting 14 amino acid residues were identified. These alleles are located in three distinct clusters in the center of the rpoB gene. We discuss the implications of our results with regards to the structure of the rifampicin binding site.     

RNA dependent RNA polymerase activity associated with the double-stranded RNA virus of Giardia lamblia.

Giardia lamblia, a parasitic protozoan, can contain a double-stranded RNA (dsRNA) virus, GLV (1). We have identified an RNA polymerase activity present specifically in cultures of GLV infected cells. This RNA polymerase activity is present in crude whole cell lysates as well as in lysates from GLV particles purified from the culture medium. The RNA polymerase has many characteristics common to other RNA polymerases (e.g. it requires divalent cations and all four ribonucleoside triphosphates), yet it is not inhibited by RNA polymerase inhibitors such as alpha-amanitin or rifampicin. The RNA polymerase activity synthesizes RNAs corresponding to one strand of the GLV genome, although under the present experimental conditions, the RNA products of the reaction are not full length viral RNAs. The in vitro products of the RNA polymerase reaction co-sediment through sucrose gradients with viral particles; and purified GLV viral particles have RNA polymerase activity. The RNA polymerase activities within and outside of infected cells closely parallel the amount of virus present during the course of viral infection. The similarities between the RNA polymerase of GLV and the polymerase associated with the dsRNA virus system of yeast are discussed.