Direct photolinkage of GTP to the vaccinia virus mRNA (guanine-7-) methyltransferase GTP methyl acceptor site.
Direct photolinkage of GTP to the vaccinia virus mRNA (guanine-7-) methyltransferase GTP methyl acceptor site.
复制标题
GTP 与痘苗病毒 mRNA (鸟嘌呤-7-) 甲基转移酶 GTP 甲基受体位点的直接光连接。
DOI：
10.1021/bi00199a011
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发表时间：
1994
期刊：
影响因子：
2.9
通讯作者：
Higman,MA
中科院分区：
文献类型：
--
作者：
Niles,EG;Christen,L;Higman,MA
Revised Manuscript Received June 17, 1994® abstract: Direct Uv photolinkage of [a-32P] GTP to the methyl acceptor site of the vaccinia virus (guanine-7-) methyltransferase was attempted in order to identify the GTP binding region of this enzyme. Lowefficiency photolinkage of GTP to the carboxyl terminal domain of the large subunit, DIR498-844, was achieved and shown to be specific by several criteria. The half-saturation value for GTP was determined to be 35 fiM which is equivalent to the catalytic Km for the substrate. GTP photolinkage was shown to be inhibited by GpppA, a substrate for the methyltransferase reaction, better than GMepppA, the reaction product. The addition of MgCl2, known to prevent GTP from serving as a methyl group acceptor in this reaction, was found to eliminate GTP photolinkage. Finally, AdoHcy, a potent product inhibitor of AdoMet binding, failed to inhibit GTPphotolinkage, demonstrating that GTP was not linked to the AdoMet binding site. Chemical cleavage of the GTP-labeled enzyme permitted the identification of multiple radioactive peptides, demonstrating the existence of multiple interaction sites in the carboxyl terminal domain of the DIR subunit. The addition of the small D12L subunit has been shown to activate the (guanine-7-) methyltransferase activity in DIR498-844 3 0-50-fold. The efficiencyof GTP photolinkage to the isolated D1R498-844 domain5 however, was found to be only marginally effected by the addition of the D12L subunit, demonstrating that this enhancement of mRNA (guanine-7-) methyltransferase activity mediated by D12L was not achieved by altering the strength of GTP binding.Vaccinia, a member of the poxvirus family, is a doublestranded DNA containing virus that conducts its replication cycle in the cytoplasm of infected cells. In order to carry on this unusual life cycle, the virus has evolved to encode many enzymes usually found in the nucleus required for viralDNA replication and gene expression (Moss, 1990). Vaccinia virus presents an unusual opportunity to apply both biochemical and genetic approaches to investigate the mechanisms of gene
