Identification of the guanylyltransferase region and active site in reovirus mRNA capping protein lambda 2

The 144-kDa lambda 2 protein of mammalian reovirus catalyzes a number of en zymatic activities in the capping of reovirus mRNA, including the transfer of GMP from GTP to the 5' end of the 5'-diphosphorylated nascent transcript , This reaction proceeds through a covalently autoguanylylated lambda 2-GMP intermediate. The smaller size of RNA capping guanylyltransferases from ot her organisms suggested that the lambda 2-associated guanylyltransferase wo uld be only a part of this protein. Limited proteinase K digestion of bacul ovirus-expressed lambda 2 was used to generate an amino-terminal M-r 42,000 fragment that appears to be both necessary and sufficient for guanylyltran sferase activity. Although lysine 226 was identified by previous biochemica l studies as the active-site residue that forms a phosphoamide bond with GM P in autoguanylylated lambda 2, mutation of lysine 226 to alanine caused on ly a partial reduction in guanylyltransferase activity at the autoguanylyla tion step. Alanine substitution for other lysines within the amino-terminal region of lambda 2 identified lysine 190 as necessary for autoguanylylatio n and lysine 171 as an important contributor to autoguanylylation. A novel active-site motif is proposed for the RNA guanylyltransferases of mammalian reoviruses and other Reoviridae members.