Inhibition of RNA Polymerase by Streptolydigin

TWO antibiotics inhibit RNA synthesis by interacting directly with RNA polymerase. The rifamycin series^1–3 inhibit before RNA chain initiation and are without apparent effect during polymerization. Streptolydigin, however, inhibits initiation and chain elongation^4–9. Using the d(A-T)-directed reaction as a model system¹⁰, we will show that streptolydigin stabilizes the polymerase-template interaction.     

Streptolydigin, an Inhibitor of Oxidative Phosphorylation in Rat Liver Mitochondria

Streptolydigin interferes with oxidative phosphorylation in rat liver mitochondria. The agent acts primarily as an uncoupler of respiration-associated phosphorylation but also impairs respiration to various degrees depending on the substrate. Streptolydigin partially inhibits electron flow at a point past the cytochrome b and prior to the cytochrome c reduction site. Streptolydigin also inhibits the function of the enzyme ribonucleic acid polymerase in whole bacterial cells and cell-free systems. The streptolydigin concentrations that cause effective inhibition of ribonucleic acid polymerase in cell-free systems are approximately 10 times less than those required to inhibit oxidative phosphorylation in mitochondria.     

Rifampicin-resistant bacteriophage PBS2 infection and RNA polymerase in Bacillus subtilis

Bacteriophage PBS2 replication is unaffected by rifampicin and other rifamycin derivatives, which are potent inhibitors of Bacillus subtilis RNA synthesis. Extracts of gently-lysed infected cells contain a DNA-dependent RNA polymerase activity which is specific for uracil-containing PBS2 DNA. The PBS2-induced RNA polymerase is insensitive to rifamycin derivatives which inhibit the host's RNA polymerase.     

Inhibition of RNA polymerase by streptolydigin: no cycling allowed

Bacterial RNA polymerase is a common target for many antibiotics. In two recent papers in Cell and Molecular Cell, and describe a structural basis for inhibition of bacterial RNA polymerase by the antibiotic streptolydigin. Streptolydigin may prevent distortion of a "bridge" alpha helix postulated to occur during the nucleotide addition cycle of RNA polymerase or may block a small movement of the bridge helix that helps load nucleotide triphosphates into the active site.     

Inhibition of isolated yeast and mycelial phase RNA polymerases of Histoplasma capsulatum by rifamycin derivatives

Various derivatives of rifamycin were shown to inhibit the RNA polymerases of the yeast and mycelial phases of Histoplasma capsulatum. The relative potency of each of the derivatives against the isolated polymerases was the same as the potency of each against the viable organism. RNA polymerase PC III from the yeast phase was more susceptible to the rifamycin derivatives than yeast phase polymerases PC I and PC II and the biggest differences in susceptibility were seen with the derivative AF/ABDP (2,6-dimethyl-4-benzyl-4-demethyl-rifamycin). The susceptibility pattern of the mycelial polymerase activity was identical to the yeast polymerase PC III.     

RNA polymerase-rifamycin. A molecular model of inhibition

By fluorimetric titration of Rifs (E. coli B) and Rifr (E. coli rpoB255) RNA polymerases with rifamycin, the mutant polymerase was demonstrated to bind rifamycin. A comparison of spatial structures of rifamycin and dinucleotide fragment of RNA in the hybrid with DNA revealed their similarity. Taking into account this structural similarity and also the fact that two phosphodiester bonds can be formed by RNA polymerase in the presence of rifamycin, a model for the inhibition mode was proposed. According to this model, rifamycin occupies the place of two terminal nucleotides of synthesized, but not translocated pentanucleotide in the transcribing complex. Asp-516 of the wild type beta-subunit was assumed to form a hydrogen bond with the rifamycin C(23) hydroxyl group. On the base of this model, reduced "cycling" synthesis of tetra-, penta-... up to decanucleotides by the Rifr RNA polymerase, in comparison with Rifs, was predicted.     

Streptovaricins inhibit Focus Formation by MSV (MLV) Complex

We recently reported that the streptovaricins inhibit the reaction by which DNA is transcribed from the RNA template resident in murine leukaemia virions (MLV)¹. The reports^{2, 3} which first indicated that this DNA polymerase is present in oncogenic RNA viruses have been confirmed and extended^4–8. The enzyme provides a mechanism whereby an RNA virus can insert stable genetic information into a host chromosome. Gallo and co-workers described an RNA dependent DNA polymerase in lymphoblastic leukaemic cells which was inhibited by N-demethylrifampicin⁹ and this antibiotic, together with a number of other rifamycin derivatives, also inhibited the oncogenic viral DNA polymerase¹⁰. Like the streptovaricins, the rifamycins are ansa macrolide antibiotics (Fig. 1).     

Inhibition of RNA polymerase from Bacillus thuringiensis and Escherichia coli by beta-exotoxin.

The characteristics of exotoxin inhibition of deoxyribonucleic acid (DNA) dependent ribonucleic acid (RNA) polymerase isolated from Escherichia coli and Bacillus thuringiensis were investigated. RNA polymerase isolated from a variety of growth stages was partially purified and assayed using several different native and synthetic DNA templates, and exotoxin inhibition patterns were recorded for each. Although 8 to 20-h RNA polymerase extracts of E. coli retained normal sensitivity to exotoxin (50% inhibition at a concentration of 7.5 X 10(-6) M exotoxin), RNA polymerase isolated from late exponential and ensuing stationary-phase cultures of B. thuringiensis were nearly 50% less sensitive than exponential RNA polymerase activity. Inhibition patterns relating culture age at the time of RNA polymerase extraction to exotoxin inhibition suggested a direct correlation between diminishing exotoxin sensitivity and sporulation. Escherichia coli RNA polymerase could be made to mimic the B. thuringiensis exotoxin inhibition pattern by removal of sigma from the holoenzyme. After passage through phosphocellulose, exotoxin inhibition of the core polymerase was 30% less than the corresponding inhibition of E. coli holoenzyme. Heterologous enzyme reconstruction and assay were not possible due to loss of activity from the B. thuringiensis preparation during phosphocellulose chromatography, apparently from the removal of magnesium. In enzyme velocity studies, inhibition with exotoxin was noncompetitive with respect to the DNA template in the RNA polymerase reaction.     

Mechanism of action of rifamazine, a member of a new class of (dimeric) rifamycins.

1. Rifamazine (AF/RP) a dimeric rifamycin, is active against bacterial DNA-dependent RNA polymerase and against viral RNA-dependent DNA polymerase. 2. Rifamazine is active also against DNA-dependent RNA polymerase extracted from rifampicin-resistant mutants of Escherichia coli. It does not interfere with enzyme-template interaction or with RNA elongation. It blocks initiation. 3. A comparison is made between the mechanism of action of rifamazine and that of rifampicin, and of AF/013 (octyloxime of 3-formylrifamycin SV), a C-class rifamycin. Our results show that the mechanism of action of rifamazine is more similar to that of rifampicin than to that of the octyloxime derivative. 4. Activity of rifamazine against RNA polymerase from rifampicin-resistant mutants is thought to be due to binding of the dimer to both the rifamycin-specific binding site and to a second weak site.     

Inclusion of polyvinylpyrrolidone in the polymerase chain reaction reverses the inhibitory effects of polyphenolic contamination of RNA.

Polysaccharides, secondary metabolites and poly-phenolics are known to co-isolate with nucleic acids from plant tissues resulting in inhibition of molecular manipulations. RNA isolated from the polyphenolic-rich resurrection plant, Myrothamnus flabellifolius, was demonstrated to inhibit a standard polymerase chain reaction used as an assay despite the inclusion of the polyphenolic-binding compound poly(1-vinylpyrrolidone-2) (PVP) into the RNA isolation medium. This inhibition was, however, reversed by the addition of PVP into the PCR mixture itself. Confirmation of the inhibitory effect of polyphenolics on PCR was obtained by addition of green tea polyphenolics to the standard PCR assay. This inhibition was also reversed by the simultaneous inclusion of PVP.     

A fluorescence polarization-based screening assay for nucleic acid polymerase elongation activity

We have devised a simple high-throughput screening compatible fluorescence polarization-based assay that can be used to detect the elongation activity of nucleic acid polymerase enzymes. The assay uses a 5' end-labeled template strand and relies on an increase in the polarization signal from the fluorescent label as it is drawn in toward the active site by the action of the enzyme. If the oligonucleotide is sufficiently short, the fluorescence polarization signal can also be used to detect binding prior to elongation activity. We refer to the nucleic acid substrate as a polymerase elongation template element (PETE) and demonstrate the utility of this PETE assay in a microtiter plate format using the RNA-dependent RNA polymerase from poliovirus to extend a self-priming hairpin RNA. The PETE assay provides an efficient method for screening compounds that may inhibit the nucleic acid binding or elongation activities of polymerases.