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.

1, IntroductionDNA-dependent RNA polymerase catalyzes polymerisation of the RNA chain from NTPs as well as the pyrophosphate exchange reaction in NTPs [ 11. The enzyme does not catalyze pyrophosphorolysis of free RNA [2,3]. In contrast, small but detectable pyrophosphorolysis of RNA from the RNA-DNA hybrid has been demonstrated [3].In our study of pause distribution along the T7 RNA chain [4,5] a model of the RNA elongation reaction has been proposed to account for the nucleotide sequence dependence of the RNA chain elongation rate. As a necessary part, occurrence of the processive pyrophosphorolysis of the nascent RNA chain is stipulated in the model. Therefore, the predicted property has been tested.Here we show that E. coli RNA polymerase is capable of catalyzing the consecutive DNA-dependent RNA pyrophosphorolysis in the presence of inorganic pyrophosphate. An active ternary complex of the enzyme with DNA and nascent RNA, Mg*+ and inorganic pyrophosphate are needed for the reaction. NTPs are low-molecular-mass (M,) products of the reaction. The rate of pyrophosphorolysis for particular nucleotides in different regions of RNA can differ by several orders of magnitude depending on the primary structure of the RNA region that undergoes pyrophosphorolysis. The rate of the reaction ranges from 1 nucleotide for 100 h per chain to 1 nucleotide for 1 min per chain.Dissociation of the ternary complexes has also been studied. The ternary complex of RNA polymerase with DNA and nascent RNA has been shown to undergo site specific dissociation. The rate of dissociation is shown to be a function of the primary structure of RNA and the direction of the reaction. Details of these experiments are described in [6,7]. Materials and methodsKinetics of RNA degradation from the native ternary complex with T7 DNA and RNA polymerase under the action of inorganic pyrophosphate were analyzed by electrophoresis of the RNA in polyacrylamide gel [6,8] and chromatography of the low-M, product on PEIcellulose. ["'PI RNA was synthesized from Al promoter of T7 DNA for 8 min at 23°C in the presence of 100 r_IM CpA and 2.5 pM of each of the 4 NTPs, one of them being labeled with the specific activity of 100-400 Ci/mmol. The active ternary complex of RNA polymerase, T7 DNA and nascent RNA was prepared, purified from NTPs after RNA synthesis (NTP-free ternary complex) and from PPi after pyrophosphorolysis (PPl-free ternary complex) using A 1.5 m agarose gel-filtration. To pyrophosphorolyze the RNA, the NTP-free ternary complex was incubated with 1 mM PPi in the presence of Mg*+ (details in [6]). The primary structure of RNA in the ternary complex was determined by means of specific termination of the RNA synthesis by 3'-0CH3-NTPs [8]. Purification and properties of RNA polymerase, DNA and other materials have been described in [4-81. Results and discussionIn this study we have used the ternary complex of RNA polymerase with DNA of T7 phage deletion mutant DIII and nascent RNA of 20-120 nucleotides long synthesized from AI promoter [6,8] (fig

It is demonstrated here that rat liver DNA polymerase /I catalyzes the pyrophosphorolysis reaction with pyrophosphate (PP<) and its analogues. The substrate specificity of the PPi-binding site of several DNA polymerases was investigated. It was discovered that the ability of DNA polymerases to utilize PPi analogues instead of PPi in the pyrophosphorolysis reaction was markedly restricted. Only imidodiphosphate and methylenediphosphonate were demonstrated as participating in this process. Oxodiphosphonate and phosphonoformate inhibited DNA synthesis, but probably not via the interaction with the PPi-binding site of DNA polymerases.

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