Influence of 5'-terminal cap structure on the initiation of translation of vaccinia virus mRNA

Abstract

The ability of methylated vaccinia virus mRNA to bind to ribosomes derived from wheat germ of rabbit reticulocyte lysates has been studied after beta elimination, to remove the 5'-terminal m7G, and after "recapping" of beta-eliminated mRNA molecules using guanylyltransferase.guanine-7-methyltransferase complex from vaccinia virions. Removal of m7G from the mRNA results in significant loss of ability to bind to ribosomes and to simulate protein synthesis in vitro. Readdition of m7G, but not of unmethylated guanosine to the 5' end results in recovery of both of these functions. To evaluate the role of 2'-O-methylation of the penultimate ribonucleoside, mRNAs containing m7G-(5')pppA- and m7G(5')pppG- as well as m7G(5')pppAm- and m7G(5')pppGm- ends were synthesized in vitro at limiting S-adenosylmethionine concentrations by vaccinia virus cores. By comparing the cap sequences of ribosome-bound and unbound mRNAs, we concluded that 2'-O-methylation has at most a minor effect compared to that of m7G upon ribosome binding under in vitro conditions. Only at high input mRNA concentrations, at which competition might occur, was there some ribodomal enrichment of mRNAs containing a specific terminal structure, namely m7G(5')pppAm-.