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

真核细胞中5‘末端帽子结构的合成至少需要4种酶活性。MRNA封顶酶催化形成封闭结构GpppN，由两种酶活性组成，即mRNA鸟苷酸转移酶(GTase)和RNA5‘-三磷酸酶(Tpase)。为了阐明mRNA封端的分子机制，我们对不同来源的封端酶的结构和功能进行了研究。还研究了封顶和转录之间的关系。动物的封帽酶由一条多肽链组成，分子量约为70,000，而酵母酶由两条独立的链组成，α链(52 KDa，GTase)和β链(80 KDa，TPase)。酵母α亚基催化的帽子形成通过酶-GMP共价反应中间体进行。中间体中的GMP被发现与酶的赖氨酸残基结合。用酵母封闭酶抗体筛选酵母基因组表达文库，获得了CEG1的编码基因。该基因以单拷贝形式存在于7号染色体上，对酵母菌的生长至关重要。CEG1在原核表达系统中得到高效表达，重组α链得到高度纯化。在没有β链的情况下，它催化帽子的形成以及酶-GMP复合体的形成。用重组酶测定了α链活性部位的氨基酸序列，包括GMP结合部位。通过使用来自HeLa细胞的体外转录系统的研究，我们证明了封闭酶与RNA聚合酶II的起始复合体特异相关。为了研究仙台病毒(一种负链RNA病毒)的mRNA封顶机制，我们用纯化的病毒粒子建立了高效、可靠的体外转录系统。我们发现了一种独特的帽子形成机制(GDP转移机制)，它不同于细胞机制(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).