PHYLOGENY OF MESSENGER-RNA CAPPING ENZYMES

The m(7)GpppN cap structure of eukaryotic mRNA is formed cotranscripti onally by the sequential action of three enzymes: RNA triphosphatase, RNA guanylyltransferase, and RNA (guanine-7)-methyltransferase. A mult ifunctional polypeptide containing all three active sites is encoded b y vaccinia virus. In contrast, fungi and Chioreiia virus encode monofu nctional guanylyltransferase polypeptides that lack triphosphatase and methyltransferase activities. Transguanylylation is a two-stage react ion involving a covalent enzyme-GMP intermediate. The active site is c omposed of six protein motifs that are conserved in order and spacing among yeast and DNA virus tapping enzymes. We performed a structure-fu nction analysis of the six motifs by targeted mutagenesis of Ceg1, the Saccharomyces cerevisiae guanylyltransferase. Essential acidic, basic , and aromatic functional groups were identified. The structural basis for covalent catalysis was illuminated by comparing the mutational re sults with the crystal structure of the Chlorella virus capping enzyme . The results also allowed us to identify the capping enzyme of Caenor habditis elegans. The 573-amino acid nematode protein consists of a C- terminal guanytyltransferase domain, which is homologous to Ceg1 and i s strictly conserved with respect to all 16 amino acids that are essen tial for Ceg1 function, and an N-terminal phosphatase domain that bear s no resemblance to the vaccinia triphosphatase domain but, instead, h as strong similarity to the superfamily of protein phosphatases that a ct via a covalent phosphocysteine intermediate.