Mechanism of mRNA capping by vaccinia virus guanylyltransferase: characterization of an enzyme--guanylate intermediate.

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Abstract
Vaccinia virus RNA guanylyltransferase catalyzes the transfer of GMP from GTP to the 5''-triphosphate or diphosphate terminus of RNA to generate the cap structure G(5'')ppp(5'')N-. The guanylylation reaction consists of a series of at least 2 partial reactions. In the 1st of these, GTP reacts with capping enzyme in the absence of an RNA acceptor to form a covalent enzyme-guanylate intermediate. The GMP is linked to the 95,000 MW subunit of the capping enzyme via a phosphoamide bond, as judged by the acid-labile, alkali-stable nature of the bond and by the susceptibility of the linkage to cleavage by hydroxylamine at pH 4.75. In the 2nd reaction, the isolated enzyme-guanylate complex transfers the guanylate moiety to triphosphate-terminated poly(A) to yield the 5'' cap structure GpppA or to PPi to regenerate GTP. Both partial reactions of transguanylylation require a divalent cation.

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