Studies on the Mechanism of Formation of mRNA 5'-Terminal Cap Structure and its Function

真核生物mRNA中5 '端帽结构的合成至少需要四种酶活性。催化封闭结构GpppN形成的mRNA加帽酶由两种酶活性组成，mRNA鸟苷酰转移酶（GT酶）和RNA 5 '-三磷酸酶（Tpase）。为了阐明mRNA加帽的分子机制，我们研究了不同来源的加帽酶的结构和功能。并对加帽与转录的关系进行了研究.动物加帽酶由Mr约为70，000的单链多肽链组成，而酵母酶由两条独立的链，α（52 KDa，GT酶）和β（80 KDa，TG酶）组成。由酵母α亚基催化的帽形成通过酶-GMP共价反应中间体进行。发现中间体中的GMP与酶的赖氨酸残基结合。通过用抗酵母加帽酶的抗体筛选酵母基因组表达文库来分离编码α亚基（CEG 1）的基因。该基因在7号染色体上以一个拷贝存在，并且对于酵母的生长是必需的。CEG 1在E.大肠杆菌中表达，重组α链得到高度纯化。在β链不存在的情况下，它催化帽形成以及酶-GMP复合物的形成。使用重组酶确定α链活性位点的氨基酸序列，包括GMP结合位点。从使用来自HeLa细胞的体外转录系统的研究中，我们证明了加帽酶与RNA聚合酶II的起始复合物特异性相关。为了研究仙台病毒（一种负链RNA病毒）mRNA加帽的机制，我们用纯化的病毒粒子建立了一个高效、可靠的体外转录系统。我们发现了一种独特的帽形成机制（GDP转移机制），这是不同于细胞的一个（GMP转移机制）。

At least four enzymatic activities are required for the synthesis of the 5'-terminal cap structure in eukaryotic mRNA. mRNA capping enzyme which catalyzes the formation of a blocked structure, GpppN, consists of two enzyme activities, the mRNA guanylyltransferase (GTase) and the RNA 5'-triphosphatase (Tpase). To elucidate the molecular mechanism of mRNA capping, we studied the structure and the function of capping enzyme from various sources. The relationship between capping and transcription was also studied.1. The animal capping enzyme consists of a single polypeptide chain with an Mr of approximately 70,000, while the yeast enzyme is composed of two separate chains, alpha (52 KDa, GTase) and beta (80 KDa, TPase). Cap formation catalyzed by yeast alpha subunit proceeds through an enzyme-GMP covalent reaction intermediate. The GMP in the intermediate was found to be bound to a lysine residue of the enzyme.2. The gene encoding the alpha subunit (CEG1) was isolated by screening a yeast genomic expression library with antibodies against yeast capping enzyme. The gene is present as one copy in chromosome 7 and is essential for the growth of yeast.3. CEG1 was expressed in E. Coli and the recombinant alpha chain was highly purified. It catalyzed cap formation as well as the enzyme-GMP complex formation in the absence of the beta chain.4. The amino acid sequence of the alpha chain active site, including the GMP binding site was determined using the recombinant enzyme.5. From studies using an in vitro transcription system derived from HeLa cells, we demonstrated that capping enzyme is specifically associated with the initiation complex of RNA polymerase II.6. To investigate the mechanism of mRNA capping in Sendai virus (a negative strand RNA virus), we developed an efficient and faithful in vitro transcription system with purified virion. We found a unique mechanism of cap formation (GDP transfer mechanism) which is different from the cellular one (GMP transfer mechanism).