Characterization of the donor and acceptor specificities of mRNA 
guanylyltransferase and mRNA (guanine-7-)-methyltransferase isolated from 
vaccinia virus cores has enabled us to discriminate between alternative reaction 
sequences leading to the formation of the 5'-terminal m7G(5')pppN-structure. The 
mRNA guanylyltransferase catalyzes the transfer of a residue of GMP from GTP to 
acceptors which possess a 5'-terminal diphosphate. A diphosphate-terminated 
polyribonucleotide is preferred to a mononucleoside diphosphate as an acceptor 
suggesting that the guanylyltransferase reaction occurs after initiation of RNA 
synthesis. Although all of the homopolyribonucleotides tested (pp(A)n, pp(G)n, 
pp(I)n, pp(U)n, and pp(C)n) are acceptors for the mRNA guanylyltransferase 
indicating lack of strict sequence specificity, those containing purines are 
preferred. Only GTP and dGTP are donors in the reaction; 7-methylguanosine (m7G) 
triphosphate specifically is not a donor indicating that guanylylation must 
precede guanine-7-methylation. The preferred acceptor of the mRNA 
(guanine-7-)-methyltransferase is the product of the guanylyltransferase 
reaction, a polyribonucleotide with the 5'-terminal sequence G(5')pppN-. The 
enzyme can also catalyze, but less efficiently methylation of the following: 
dinucleoside triphosphates with the structure G(5')pppN, GTP, dGTP, ITP, GDP, 
GMP, and guanosine. The enzyme will not catalyze the transfer of methyl groups 
to ATP, XTP, CTP, UTP, or to guanosine-containing compounds with phosphate 
groups in either positions 2' or 3' or in 3'-5' phosphodiester linkages. The 
latter specificity provides an explanation for the absence of internal 
7-methylguanosine in mRNA. In the presence of PPi, the mRNA guanylyltransferase 
catalyzes the pyrophosphorolysis of the dinucleoside triphosphate G(5')pppA, but 
not of m7G(5')pppA. Since PPi is generated in the process of RNA chain 
elongation, stabilization of the 5'-terminal sequences of mRNA is afforded by 
guanine-7-methylation.