Isolation and Characterization of the Yeast mRNA Capping Enzyme β Subunit Gene Encoding RNA 5′-Triphosphatase, Which Is Essential for Cell Viability

Abstract

The yeastSaccharomyces cerevisiaemRNA capping enzyme is composed of two subunits of α (52 kDa, mRNA guanylyltransferase) and β (80 kDa, RNA 5′-triphosphatase). We have isolated the α subunit gene (CEG1) by immunological screening. In this report, with the aid of partial amino acid sequences of purified yeast capping enzyme, we isolated the gene, designatedCET1,encoding theS. cerevisiaecapping enzyme β subunit. Amino acid sequence analysis revealed that the gene encodes for 549 amino acids with a calculatedMrof 61,800 which is unexpectedly smaller than the size estimated by SDS-PAGE. Gene disruption experiment showed thatCET1is essential for yeast cell growth. The purified recombinantCET1gene product, Cet1, exhibited an RNA 5′-triphosphatase activity which specifically removed the γ-phosphate from the triphosphate-terminated RNA substrate, but not from nucleoside triphosphates, confirming the identity of the gene. Interaction between the Cet1 and the Ceg1 was also studied by the West-Western procedure using recombinant Ceg1-[32P]GMP as probe.