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首页> 外文期刊>Molecular and Cellular Biology >Mutational analysis of mRNA capping enzyme identifies amino acids involved in GTP binding, enzyme-guanylate formation, and GMP transfer to RNA.
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Mutational analysis of mRNA capping enzyme identifies amino acids involved in GTP binding, enzyme-guanylate formation, and GMP transfer to RNA.

机译：对mRNA封端酶的突变分析可确定参与GTP结合，酶鸟苷酸形成和GMP向RNA转移的氨基酸。

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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.
机译：牛痘病毒mRNA封端酶是一种多功能蛋白，具有RNA三磷酸酶，RNA鸟苷基转移酶，RNA（鸟嘌呤7）甲基转移酶和转录终止因子活性。该蛋白是分别由痘苗病毒D1和D12基因编码的95 kDa和33 kDa亚基的异二聚体。加帽反应需要通过共价酶-（赖氨酰-GMP）中间体将GMP从GTP转移到mRNA的5'-二磷酸端。活性位点位于序列元素KxDG（基序I）内D1亚基的Lys-260处，该位点在酵母和其他DNA病毒的加帽酶中保守，并在RNA和DNA连接酶的共价腺苷酸化的活性位点保守。四个额外的序列基序（II至V）以相同的顺序保存，并且在加帽酶和几种ATP依赖的连接酶之间具有相似的间隔。通过牛痘病毒D1蛋白的突变分析，解决了这些常见序列元素与RNA封端反应的相关性。九个丙氨酸取代突变针对II至V的基序。突变的D1多肽的组氨酸标签版本与D12亚基在细菌中共表达，并通过Ni亲和力和磷酸纤维素层析步骤纯化了带有His标签的异二聚体。每种突变酶均保留了三磷酸酶，甲基转移酶和终止因子的活性，而九种突变酶中的六种在转鸟苷酸化的某些方面存在缺陷。图案III，IV和V中的单个突变对酶对GTP的亲和力，共价催化速率（EpG形成）或GMP从酶向RNA的转移具有独特的影响。这些结果与酵母RNA封端酶的突变研究一致，并提出了共价核苷酸转移的保守结构基础。

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