Inhibitory effect of methylated derivatives of guanylic acid for protein synthesis with reference to the functional structure of the 5′-‘cap’ in viral messenger RNA

Abstract

Guanylic acid modified variously with methyl groups on base or sugar moieties were synthesized chemically and their inhibitory effects on protein synthesis were tested in a wheat germ cell-free system using mRNAs from cytoplasmic polyhedrosis virus and tobacco mosaic virus. The confronting dinucleotide m 7G 5′ pppA that corresponds to the most simple ‘cap’ structure of an eukaryotic mRNA is a strong inhibitor of protein synthesis, but non-methylated G 5′ pppA or G 5′ ppA is not inhibitory. The strong inhibitory effect is observed only by 7-methylguanylic acid (pm 7G). Among 11 derivatives of pG, the most effective inhibitors are methylated at the 7-position. Further methylation at the other position sometimes cancels the inhibitory effect. Although pm 7G carries a positively charged base, other nucleotides which carry a plus charged base (1-methyladenylic acid and 2-methylthio-7-methylinosinic acid) were not inhibitory. Thus, methylation at the 7-position on guanylic acid is specifically required for the inhibitory effect. Addition of pm 7G was inhibitory for the formation of the initiation complex for eukaryotic protein synthesis. These results suggest that the ‘cap’ component containing 7-methylguanylic acid in viral mRNA participates during protein synthesis, especially in its initial steps. Protein synthesis in a bacterial cell-free system was not inhibited by addition of m 7GpppA or pm 7G when either TMV RNA or phage MS2 RNA was used as an mRNA.