Structure and function of mRNA capping enzyme system

mRNA帽结构是由mRNA加帽酶（RNA 5 '-三磷酸酶和mRNA鸟苷酰转移酶）和mRNA（鸟嘌呤-7-）甲基转移酶（mRNA帽甲基转移酶）催化的一系列反应合成的。在此，我们分离了酿酒酵母RNA 5 '-三磷酸酶（加帽酶β亚基）基因（CET 1）、两种人加帽酶cDNA（hCAP 1a、B）和三种人mRNA（鸟嘌呤-7-）甲基转移酶cDNA（hCMT 1a、B、c）。CET 1编码549个氨基酸，计算的Mr为61，849，并且与人加帽酶（hCAP 1）的RNA 5 '-三磷酸酶（包括酪氨酸特异性蛋白磷酸酶（PTP）活性位点基序）没有显著的序列相似性。基因破坏实验表明，CET 1是酵母细胞生长所必需的。hCAP 1a和hCAP 1b分别编码597和541个氨基酸，并且仅在编码酶的C-末端部分的区域不同。在hCAP 1中观察到mRNA鸟苷酸转移酶之间保守的区域，除了 ...更多信息 hCAP 1b蛋白中缺少一个保守区。hCAP 1a缺失突变体分析表明，N端213个氨基酸的PTP基序片段具有RNA 5 '-三磷酸酶活性，C端369个氨基酸的鸟苷酰转移酶活性。hCAP 1b显示RNA 5 ′-三磷酸酶活性，但未检测到酶-GMP共价复合物形成或帽结构形成。hCMT 1a和hCMT 1b分别编码476和504个氨基酸，并且仅在编码氨基酸残基465之后的酶的C-末端部分的区域不同。RT-PCR结果显示，3种mRNA在各组织中均有表达。与其他病毒和细胞酶的氨基酸序列的比较，推导出的氨基酸序列之间的mRNA（鸟嘌呤-7-）甲基转移酶的高度保守的区域。使用在细菌中表达的重组蛋白分析了hCAP 1a、hCMT 1a和RNA聚合酶II的相互作用，并且清楚地显示了它们相互作用所必需的位置。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