MUTATIONAL ANALYSIS OF YEAST MESSENGER-RNA CAPPING ENZYME

RNA guanylyltransferase (capping enzyme) catalyzes the transfer of GMP from GTP to the 5'-diphosphate end of mRNA. The capping reaction proc eeds via an enzyme-guanylate intermediate in which GMP is linked coval ently to a lysine residue of the enzyme. In the capping enzyme of Sacc haromyces cerevisiae, GMP is attached to a 52-kDa polypeptide, identif ied as the product of the essential CEG1 gene. The amino acid sequence of the CEG1 protein includes a moth, Lys(70)-Thr-Asp-Gly, that is con served at the active site of vaccinia virus RNA guanylyltransferase an d which is similar to the KXDG sequence found at the active sites of R NA and]DNA ligases. To evaluate the role of this moth in the function of the yeast enzyme, we have expressed the CEG1 protein in active form in Escherichia coli. Replacement of Lys(70) Or Gly(73) With alanine a brogated enzyme-guanylate formation in vitro; in contrast, alanine sub stitutions at Thr(71) Or ASp(72) merely reduced activity relative to w ild-type enzyme. The K70A and G73A mutations were lethal to yeast, whe reas yeast carrying the T71A and D72A alleles of CEG1 were viable. The se results implicate Lys(70) as the active site of yeast guanylyltrans ferase and provide evidence that cap formation per se is an essential function in eukaryotic cells.