Formation of the guanylylated and methylated 5′-terminus of vaccinia virus mRNA

Abstract mRNA was synthesized in vitro by vaccinia virus particles in the presence of S-adenosyl-[methyl-3H]methionine and α- or β,γ-32P-labeled ATP or GTP. The two 5′-terminal structures m7G(5′)ppp(5′)Gm and m7G(5′)ppp(5′)Am were isolated after P1 nuclease digestion of the RNA. The distribution of radioactivity between the methylated mononucleotides and inorganic phosphate released by nucleotide pyrophosphatase treatment of m7G(5′)ppp(5′)Gm and m7G(5′)ppp(5′)Am was consistent with the formation of the latter structures by condensation of pG residues from GTP with ppG- and ppA- at the 5′-termini of vaccinia mRNAs. An alternative mechanism involving the transfer of ppG residues to pA- at the 5′-terminus of the RNA was ruled out by the 32P-labeling data as well as by the formation of m7G(5′)ppp(5′)Am- when β,γ-methylene-GTP was substituted for GTP. At limiting concentrations of S-adenosylmethionine, the predominant methylated 5′-terminal structure was m7G(5′)ppp(5′)N- in which the penultimate nucleoside was unmethylated; in the absence of S-adenosylmethionine, G(5′)ppp(5′)N- as well as unblocked ppN- termini were detected. On the basis of the structures formed under various conditions, the following sequence of reactions was considered: γβα ppG + β′α′ ppN −→ G (5′) N −+ γβ PPi G (5′) ppp (5′) N −+ AdoMet → m 7 G (5′) ppp (5′) N −+ AdoHcy m 7 G (5′) ppp (5′) N −+ AdoMet → m 7 G (5′) ppp (5′) N m + AdoHcy