Cong P, Shuman S
Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA.
Mol Cell Biol. 1995 Nov;15(11):6222-31. doi: 10.1128/MCB.15.11.6222.
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.
痘苗病毒mRNA加帽酶是一种多功能蛋白，具有RNA三磷酸酶、RNA鸟苷酰转移酶、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亚基组成的异二聚体。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'-二磷酸末端。活性位点位于D1亚基的Lys-260处，在一个序列元件KxDG（基序I）内，该元件在酵母和其他DNA病毒的加帽酶以及RNA和DNA连接酶的共价腺苷酸化活性位点中是保守的。另外四个序列基序（II至V）在加帽酶和几种ATP依赖性连接酶中以相同顺序和相似间隔保守。通过对痘苗病毒D1蛋白的突变分析探讨了这些共同序列元件与RNA加帽反应的相关性。九个丙氨酸取代突变靶向基序II至V。突变的D1多肽的组氨酸标记版本在细菌中与D12亚基共表达，并且通过镍亲和层析和磷酸纤维素层析步骤纯化带His标签的异二聚体。虽然每个突变酶都保留了三磷酸酶、甲基转移酶和终止因子活性，但九个突变酶中有六个在转鸟苷酰化的某些方面存在缺陷。基序III、IV和V中的单个突变对酶与GTP的亲和力、共价催化速率（EpG形成）或GMP从酶向RNA的转移有独特影响。这些结果与酵母RNA加帽酶的突变研究一致，并表明共价核苷酸转移存在保守的结构基础。
