Abstract
Capping of reovirus mRNAs is catalyzed by a guanylyltransferase that corresponds to virion structural polypeptide lambda 2. It forms a phosphoamide linked enzyme-pG covalent complex as an intermediate in the capping reaction. The nucleotide attachment site on lambda 2 was localized to a region between amino acids 213 and 269 by incubating virus particles with [alpha-32P]GTP followed by proteolytic cleavage and analysis of the resulting fragments using sequence-directed antibodies as probes. The 213-269 region contains as potential GMP acceptors a single lysine, 1 arginine, and 4 histidine residues, as deduced from the nucleotide sequence of the L2 gene encoding lambda 2. Digestion of 32P-labeled capping intermediate with alkali after oxidation and beta-elimination yielded phospholysine as the only phosphoamino acid, localizing the active site to a region in lambda 2 that includes the lysine at position 226.
Highlights
Active Site Localization in a Viral mRNA Capping Enzyme*From the Center for Advanced Biotechnology and Medicine, Piscatawuy, New Jersey 08854-5638
Capping of reovirus mRNAs is catalzyed by a guanylyltransferase that corresponds to virion structural polypeptide X2
This reaction proceeds in two distinct steps, i.e. formation of an enzyme-GMP intermediate followed by nucleotide transfer and recycling of guanylyltransferase: E + guanosine [a-3zP]triphosphate (GTP) - E.pG + PPi
Summary
From the Center for Advanced Biotechnology and Medicine, Piscatawuy, New Jersey 08854-5638. Capping of reovirus mRNAs is catalzyed by a guanylyltransferase that corresponds to virion structural polypeptide X2. It forms a phosphoamide linked enzyme-pG covalent complex as an intermediate in the capping reaction. This reaction proceeds in two distinct steps, i.e. formation of an enzyme-GMP intermediate followed by nucleotide transfer and recycling of guanylyltransferase:. Autoradiography, as shown initially for the capping enzyme in vaccinia virus [17] By this same procedure, the reovirus mRNA guanylyltransferase has been identified as polypeptide X2 [18, 19], a 167-kDa structural subunit of pentameric spikes that extend from the core to the outer surface of virions [20]. On the basis of this information, we have prepared and used peptide sequencedirected antibodies to establish that in reovirus serotype 3, GMP is attached to the mRNA guanylyltransferase at a single lysine at position 226 in polypeptide X2
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