Vaccinia Virus mRNA (Guanine-7-)Methyltransferase: Mutational Effects on Cap Methylation and AdoHcy-Dependent Photo-Cross-Linking of the Cap to the Methyl Acceptor Site

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• 作者：Xiangdong Mao and Stewart Shuman
• 刊名：Biochemistry
• 出版年：1996
• 出版时间：May 28, 1996
• 年：1996
• 卷：35
• 期：21
• 页码：6900 - 6910
• 全文大小：553K
• 年卷期：v.35,no.21(May 28, 1996)
• ISSN：1520-4995

文摘

The (guanine-7-) methyltransferase domain of the vaccinia virusmRNA capping enzyme iscomposed of the C-terminal portion of the D1 subunit,D1(498-844), heterodimerized with the D12protein. In order to identify protein structural elements involvedin cap methylation, we introduced eightalanine substitution mutations within two sequence motifs ofD1(498-844)-(594)VLAIDFGNG(602)and (681)IHYSF(685)-that are conserved in the capmethyltransferase from yeast. The D1(498-844)-Ala proteins were coexpressed in bacteria with the D12 subunit, and therecombinant D1(498-844)/D12heterodimers were purified. Alanine substitutions at fivepositions-Asp-598, Gly-602, Ile-681, Ser-684,and Phe-685-had little or no effect on methyltransferase activity.Mutations at three conserved residueswere deleterious. Alanine substitution at Gly-600 reduced thespecific activity to 4% of that of the wild-type protein. Substitutions at His-682 and Tyr-683 reduced activityto 4% and 0.05%, respectively. Byfurther mutating Tyr-683 to Phe and Ser, we established that thearomatic group was essential for capmethylation, whereas the hydroxyl moiety was dispensable. Specificbinding of the methyltransferase tothe RNA cap was demonstrated by UV cross-linking to[³²P]GMP-labeled capped poly(A). Labeltransferoccurred exclusively to the D1(498-844) subunit and was competedby the cap analogs GpppA andm7GpppA. Cap-specific cross-linking to m7GpppA(pA)n wasstimulated by AdoHcy, whereas cross-linking to GpppA(pA)n was unaffected by AdoHcy, but stimulated byAdoMet. We suggest that occupancyof the methyl donor site either enhances the affinity for the capguanosine or alters the protein interfaceso that a photoreactive moiety is brought closer to the cap structure.The catalytically defective H682A,Y683A, and Y683S mutant methyltransferases were unable to cross-link tothe cap in the presence ofAdoHcy. The catalytically defective G600A mutant did cross-link tothe cap in the presence of AdoHcy,suggesting that this mutation affects the chemical step oftransmethylation.