Location of the S-adenosyl-L-methionine binding region of the vaccinia virus mRNA (guanine-7-)methyltransferase.
Location of the S-adenosyl-L-methionine binding region of the vaccinia virus mRNA (guanine-7-)methyltransferase.
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发表时间：
1994-05
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通讯作者：
M. Higman;E. G. Niles
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作者：
M. Higman;E. G. Niles
先前将异二聚体牛痘病毒mRNA加帽酶的mRNA（鸟嘌呤-7-）甲基转移酶活性定位于大亚基D1 R的羧基末端396个氨基酸。这种活性通过小亚基D12 L（Higman，M.一、克里森湖一、和奈尔斯，E. G.（1994）J.Biol.Chem.269，14974-14981）。紫外线照射特异性地将S-[甲基-3H] S-腺苷-L-甲硫氨酸与完整加帽酶和甲基转移酶亚结构域的大亚基光链接，所述甲基转移酶亚结构域由与D1 R亚基的羧基末端346个氨基酸相关的D12 L亚基组成。连接的程度被证明是依赖于孵育的长度，紫外光的强度，和活性酶和底物的浓度。共价修饰被S-腺苷-L-同型半胱氨酸抑制，S-腺苷-L-同型半胱氨酸是一种已知的mRNA（鸟嘌呤-7-）甲基转移酶活性的竞争性抑制剂，表明活性位点的特异性光标记。序列特异性化学切割的光连接的甲基转移酶结构域与温和的酸或溴化氰揭示连接的S-腺苷-L-蛋氨酸的大亚基的两个区域。溴化氰和羟胺裂解的产物的分析映射的光链接片段的氨基酸499至579和806至844的D1 R亚基。D1 R498 -844的光连接显示不受小亚基缔合的影响。
The mRNA (guanine-7-)methyltransferase activity of the heterodimeric vaccinia virus mRNA capping enzyme was previously mapped to the carboxyl-terminal 396 amino acids of the large subunit, D1R. This activity is enhanced 30- to 50-fold by the association of the the small subunit, D12L (Higman, M. A., Christen, L. A., and Niles, E. G. (1994) J. Biol. Chem. 269, 14974-14981). Irradiation with ultraviolet light specifically photolinks S-[methyl-3H]S-adenosyl-L-methionine to the large subunit of both the intact capping enzyme and a methyltransferase subdomain, which consists of the D12L subunit associated with the carboxyl-terminal 346 amino acids of the D1R subunit. The extent of linkage was shown to be dependent on the length of incubation, intensity of ultraviolet light, and the concentration of both active enzyme and substrate. The covalent modification was inhibited by S-adenosyl-L-homocysteine, a known competitive inhibitor of the mRNA (guanine-7-)methyltransferase activity, demonstrating specific photolabeling of the active site. Sequence-specific chemical cleavage of the photolinked methyltransferase domain with mild acid or cyanogen bromide revealed linkage of S-adenosyl-L-methionine to two regions of the large subunit. Analysis of the products of cyanogen bromide and hydroxylamine cleavage mapped the photolinked fragments to amino acids 499 to 579 and 806 to 844 of the D1R subunit. Photolinkage of AdoMet to D1R498-844 was shown to be unaffected by the association of the small subunit.