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

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Abstract
DNA-directed RNA polymerase from Escherichia coli can break down RNA by catalyzing the reverse of the reaction: NTP + (RNA)n = RNAn+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. 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 MWr 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 and Khomutov for phosphonoacetic acid (H2O3P-CH2-CO2H). This is explained by instability of the analogs 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. 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.