The mechanism of pyrophosphorolysis of RNA by RNA polymerase. Endowment of RNA polymerase with artificial exonuclease activity.

DNA-directed RNA polymerase from Escherichia coli can break down RNA by catalysing the reverse of the reaction: NTP + (RNA)n = (RNA)n+1 + PPi where n indicates the number of nucleotide residues in the RNA molecule, to yield nucleoside triphosphates. This reaction requires the ternary complex of the polymerase with template DNA and the RNA that it has synthesized. It is now shown that methylenebis(arsonic acid) [CH2(AsO3H2)2], arsonomethylphosphonic acid (H2O3As-CH2-PO3H2) and arsonoacetic acid (H2O3As-CH2-CO2H) can replace pyrophosphate in this reaction. When they do so, the low-Mr products of the reaction prove to be nucleoside 5'-phosphates, so that the arsenical compounds endow the polymerase with an artificial exonuclease activity, an effect previously found by Rozovskaya, Chenchik, Tarusova, Bibilashvili & Khomutov [(1981) Mol. Biol. (Moscow) 15, 636-652] for phosphonoacetic acid (H2O3P-CH2-CO2H). This is explained by instability of the analogues of nucleoside triphosphates believed to be the initial products. Specificity of recognition of pyrophosphate is discussed in terms of the sites, beta and gamma, for the -PO3H2 groups of pyrophosphate that will yield P-beta and P-gamma of the nascent nucleoside triphosphate. Site gamma can accept -AsO3H2 in place of -PO3H2, but less well; site beta can accept both, and also -CO2H. We suggest that partial transfer of an Mg2+ ion from the attacking pyrophosphate to the phosphate of the internucleotide bond of the RNA may increase the nucleophilic reactivity of the pyrophosphate and the electrophilicity of the diester, so that the reaction is assisted.     

6S RNA regulates E. coli RNA polymerase activity

The E. coli 6S RNA was discovered more than three decades ago, yet its function has remained elusive. Here, we demonstrate that 6S RNA associates with RNA polymerase in a highly specific and efficient manner. UV crosslinking experiments revealed that 6S RNA directly contacts the sigma70 and beta/beta' subunits of RNA polymerase. 6S RNA accumulates as cells reach the stationary phase of growth and mediates growth phase-specific changes in RNA polymerase. Stable association between sigma70 and core RNA polymerase in extracts is only observed in the presence of 6S RNA. We show 6S RNA represses expression from a sigma70-dependent promoter during stationary phase. Our results suggest that the interaction of 6S RNA with RNA polymerase modulates sigma70-holoenzyme activity.     

RNA polymerase activity in bovine spermatozoa

Washed mature spermatozoa from bulls incorporate ribonucleoside triphosphates into RNA using an endogenous template. Maximum incorporation was observed at 31 degrees C in the presence of MgCl2, all four ribonucleoside triphosphates, beta-mercaptoethanol, and glycine sodium hydroxide buffer at pH 9.0. The amount of synthesis was linearly dependent upon the concentration of spermatozoa and continued for at least 4 h. Digestion studies revealed the RNA to be present in a protected (intracellular?) location in the spermatozoa. The RNA synthesis was inhibited by ethidium bromide, rifampicin, acriflavine, actinomycin D, and caffeine, but not by alpha-amanitine or rifamycin SV. Fractionation of the spermatozoa by sonication and separation of the heads and tails by centrifugation through a discontinuous gradient revealed that more than half of the total RNA polymerase activity was associated with the tail fraction.     

A continuous nonradioactive assay for RNA-dependent RNA polymerase activity

Current assays for the activity of viral RNA-dependent RNA polymerases (RdRps) are inherently end-point measurements, often requiring the use of radiolabeled or chemically modified nucleotides to detect reaction products. In an effort to improve the characterization of polymerases that are essential to the life cycle of RNA viruses and develop antiviral therapies that target these enzymes, a continuous nonradioactive assay was developed to monitor the activity of RdRps by measuring the release of pyrophosphate (PP(i)) generated during nascent strand synthesis. A coupled-enzyme assay method based on the chemiluminescent detection of PP(i), using ATP sulfurylase and firefly luciferase, was adapted to monitor poliovirus 3D polymerase (3D(pol)) and the hepatitis C virus nonstructural protein 5B (NS5B) RdRp reactions. Light production was dependent on RdRp and sensitive to the concentration of oligonucleotide primer directing RNA synthesis. The assay system was found to be amenable to sensitive kinetic studies of RdRps, requiring only 6nM 3D(pol) to obtain a reliable estimate of the initial velocity in as little as 4 min. The assay can immediately accommodate the use of both homopolymer and heteropolymer RNA templates lacking uridylates and can be adapted to RNA templates containing uridine by substituting alpha-thio ATP for ATP. The low background signal produced by other NTPs can be corrected from no enzyme (RdRp) controls. The effect of RdRp/RNA template preincubation was assessed using NS5B and a homopolymer RNA template and a time-dependent increase of RdRp activity was observed. Progress curves for a chain terminator (3(')-deoxyguanosine 5(')-triphosphate) and an allosteric NS5B inhibitor demonstrated the predicted time- and dose-dependent reductions in signal. This assay should facilitate detailed kinetic studies of RdRps and their potential inhibitors using either standard or single-nucleotide approaches.     

RNA polymerase activity in germinating onion seeds

Frozen sections of endosperm cut from dry unimbibed onion seed were immersed in an aqueous solution of tritium labelled triphosphate; nucleolar RNA polymerase (ribonucleoside triphosphate: RNA nucleotidyltransferase E.C. 2.7.7.6) activity was detected by autoradiography after soaking for 10–15 min in the solution of the radioactive nucleotide. Throughout germination, activity appears to be mainly confined to the nucleolus with chromatin incorporation being very low or non-existent. In the embryo, in contrast to the endosperm, chromatin activity is initiated after 1 hr presoaking, while the nucleolus displays a lag of several hours. No incorporation could be detected in vivo before 18 hr.     

Small reovirus-specific particle with polycytidylate-dependent RNA polymerase activity.

We previously reported that virus-specific particles with polycytidylate [poly(C)]-dependent RNA polymerase activity accumulated at 30 degrees C in reovirus-infected cells. These particles sedimented heterogeneously from 300 to 550S and traversed through a 40% glycerol cushion to the pellet in 3 h at 190,000 x g. In the present report, we found that smaller particles with poly(C)-dependent RNA polymerase activity remained in the glycerol cushion. These smaller, enzymatically active particles, when purified, sedimented at 15 to 1S. They were spherical or triangular with a diameter of 11 to 12 nm. They were comprised mostly, and likely solely, of one reovirus protein, sigma NS. No particles with poly(C)-dependent RNA polymerase activity were found in mock-infected cells. Chromatography on the cation exchanger, CM-Sephadex, ascertained that sigma NS was the poly(C)-dependent RNA polymerase and showed its existence in two forms. In one form, it was enzymatically active and eluted from the column at 0.5 M KCl. In the enzymatically inactive state, it did not bind to the column. Our results suggest that the enzymatically active form of sigma NS carries a greater net positive charge than the inactive form. They also suggest that both forms of sigma NS are associated with a particle which has poly(C)-dependent RNA polymerase activity.     

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.