Structure and function of mRNA capping enzyme system

信使核糖核酸帽子结构是由信使核糖核酸封端酶(RNA5‘-三磷酸酶和信使核糖基转移酶)和信使核糖核酸(鸟嘌呤-7-)甲基转移酶(信使核糖核酸帽甲基转移酶)催化的一系列反应合成的。在这里，我们克隆了酿酒酵母RNA5‘-三磷酸酶(封闭酶β亚基)基因(CET1)、两个人类封闭酶cDNAs(hCAP1a，b)和三个人mRNA(鸟氨酸-7-)甲基转移酶cDNAs(hCMT1a，b，c)。CET1编码549个氨基酸，Mr为61,849，与人封顶酶RNA5‘-三磷酸酶(HCAP1)没有显著的序列相似性，包括酪氨酸特异性蛋白磷酸酶(PTP)活性中心基序。基因阻断实验表明，CET1是酵母细胞生长所必需的。HCAP1a和hCAP1b分别编码597和541个氨基酸，仅在酶的C末端编码区域不同。除…外，在hCAP1中还观察到了mRNADNA转移酶之间的保守区HCAP1b蛋白中缺少一个以上的保守区。HCAP1a缺失突变分析表明，含有PTP基序的N端213位氨基酸片段具有催化RNA5‘-三磷酸酶活性的作用，C端369位氨基酸片段含有mRNA鸟苷酸转移酶活性。HCAP1b具有RNA5‘-三磷酸酶活性，但没有检测到酶-GMP共价复合体的形成和帽子结构的形成。HCMT1a和hCMT1b分别编码476和504个氨基酸，仅在编码465个氨基酸残基后的酶的C末端部分有所不同。RT-PCR结果显示，3种类型的mRNAs在所有受检组织中均有表达。将推导出的氨基酸序列与其他病毒和细胞酶的氨基酸序列进行比较，发现在mRNA(鸟氨酸-7-)甲基转移酶中高度保守的区域。利用在细菌中表达的重组蛋白分析了hCAP1a、hCMT1a和RNA聚合酶II之间的相互作用，清楚地显示了它们相互作用所必需的位置。较少

The mRNA cap structure is synthesized by a series of reactions catalyzed by mRNA capping enzyme (RNA 5'-triphosphatase and mRNA guanylyltransferase) and mRNA (guanine-7-)methyltransferase (mRNA cap methyltransferase). Here we isolated Saccharomyces cerevisiae RNA 5'-triphosphatase (capping enzyme beta subunit) gene (CET1), two human capping enzyme cDNAs (hCAP1a, b), and three human mRNA (guanine-7-)methyltransferase cDNAs (hCMT1a, b, c). CET1 encodes 549 amino acids with a calculated Mr of 61, 849 and bears no significant sequence similarities to RNA 5'-triphosphatase of human capping enzyme (hCAP1) including the tyrosine specific protein phosphatase (PTP) active site motif. Gene disruption experiment showed that CET1 is essential for yeast cell growth. hCAP1a and hCAP1b encode 597 and 541 amino acids, respectively, and are different only at the region coding for the C-terminal portion of the enzyme. The regions conserved among mRNA guanylytransferases are observed in hCAP1 except that … More one conserved region was absent in the hCAP1b protein. Deletion mutant analysis of hCAP1a showed that the N-terminal 213 amino acid fragment containing PTP motif catalyzed the RNA 5'-trihosphatase activity and the C-terminal 369 amino acid fragment contained the mRNA guanylyltransferase activity. hCAP1b showed RNA 5'-triphosphatase activity, but neither enzyme-GMP covalent complex formation or cap structure formation was detected. hCMT1a and hCMT1b encode 476 and 504 amino acids, respectively, and differ only at the region coding for the C-terminal portion of the enzyme after amino acid residue 465. RT-PCR showed that all 3 types of mRNAs were expressed in every tissue examined. Comparison of the deduced amino acid sequences with those of other viral and cellular enzymes showed the regions which are highly conserved among mRNA (guanine-7-)methyltransferases. Interactions of hCAP1a, hCMT1a and RNA polymerase II were analyzed using recombinant proeins expressed in bacteria and location indispensable for their interactions were clearly shown. Less