ISOLATION AND CHARACTERIZATION OF THE YEAST MESSENGER-RNA CAPPING ENZYME BETA-SUBUNIT GENE ENCODING RNA 5'-TRIPHOSPHATASE, WHICH IS ESSENTIAL FOR CELL VIABILITY

The yeast Saccharomyces cerevisiae mRNA capping enzyme is composed of two subunits of alpha (52 kDa, mRNA guanylyltransferase) and beta (80 kDa, RNA 5'-triphosphatase). We have isolated the alpha subunit gene ( CEG1) by immunological screening. In this report, with the aid of part ial amino acid sequences of purified yeast capping enzyme, we isolated the gene, designated CET1, encoding the S. cerevisiae capping enzyme beta subunit. Amino acid sequence analysis revealed that the gene enco des for 549 amino acids with a calculated M-r of 61,800 which is unexp ectedly smaller than the size estimated by SDS-PAGE. Gene disruption e xperiment showed that CET1 is essential for yeast cell growth. The pur ified recombinant CET1 gene product, Cet1, exhibited an RNA 5'-triphos phatase activity which specifically removed the gamma-phosphate from t he triphosphate-terminated RNA substrate, but not from nucleoside trip hosphates, confirming the identity of the gene. Interaction between th e Cet1 and the Ceg1 was also studied by the West-Western procedure usi ng recombinant Ceg1-[P-32]GMP as probe. (C) 1997 Academic Press.