Mutational analysis of mRNA capping enzyme identifies amino acids involved in GTP binding, enzyme-guanylate formation, and GMP transfer to RNA.
Vaccinia virus mRNA capping enzyme is a multifunctional protein with RNA triphosphatase, RNA guanylyltransferase, RNA (guanine-7) methyltransferase, and transcription termination factor activities. The protein is a heterodimer of 95- and 33-kDa subunits encoded by the vaccinia virus D1 and D12 genes, respectively. The capping reaction entails transfer of GMP from GTP to the 5'-diphosphate end of mRNA via a covalent enzyme-(lysyl-GMP) intermediate. The active site is situated at Lys-260 of the D1 subunit within a sequence element, KxDG (motif I), that is conserved in the capping enzymes from yeasts and other DNA viruses and at the active sites of covalent adenylylation of RNA and DNA ligases. Four additional sequence motifs (II to V) are conserved in the same order and with similar spacing among the capping enzymes and several ATP-dependent ligases. The relevance of these common sequence elements to the RNA capping reaction was addressed by mutational analysis of the vaccinia virus D1 protein. Nine alanine substitution mutations were targeted to motifs II to V. Histidine-tagged versions of the mutated D1 polypeptide were coexpressed in bacteria with the D12 subunit, and the His-tagged heterodimers were purified by Ni affinity and phosphocellulose chromatography steps. Whereas each of the mutated enzymes retained triphosphatase, methyltransferase, and termination factor activities, six of nine mutant enzymes were defective in some aspect of transguanylylation. Individual mutations in motifs III, IV, and V had distinctive effects on the affinity of enzyme for GTP, the rate of covalent catalysis (EpG formation), or the transfer of GMP from enzyme to RNA. These results are concordant with mutational studies of yeast RNA capping enzyme and suggest a conserved structural basis for covalent nucleotidyl transfer.
Cong
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Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA.
Shuman
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D000595:Amino Acid Sequence; D019204:GTP-Binding Proteins / Q000737:chemistry*; D006157:Guanosine Monophosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006358:Hot Temperature; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009713:Nucleotidyltransferases / Q000737:chemistry* / Q000378:metabolism; D011489:Protein Denaturation; D012313:RNA / Q000378:metabolism; D012315:RNA Caps / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D014158:Transcription, Genetic; D014616:Vaccinia virus / Q000201:enzymology*; D000098556:mRNA Guanylyltransferases
D012315:RNA Caps; D012313:RNA; D006157:Guanosine Monophosphate; D006160:Guanosine Triphosphate; D009713:Nucleotidyltransferases; D000098556:mRNA Guanylyltransferases; D019204:GTP-Binding Proteins