MRNA CAPPING ENZYME

mRNA经历了几个基本的修饰，其中第一个是添加一个7-甲基鸟苷帽。由于帽盖在转录起始后不久发生，并且是RNA聚合酶II转录物特异性的，我们假设帽盖和转录机制在物理上或功能上相关。利用生物化学和遗传技术的结合，我们发现封盖酶通过与RNA聚合酶II的磷酸化c端结构域（CTD）特异性相互作用而被招募到起始复合物中。我们将扩展这些观察来绘制CTD和capping酶的相互作用域，确定哪些激酶(s)负责，CTD结合如何影响capping活性，以及在转录复合体中何时释放capping酶。酵母盖帽酶由两种蛋白质组成：三磷酸酶和鸟苷基转移酶。相比之下，高级真核生物的盖帽酶由具有两种活性的单一蛋白质组成。酵母三磷酸酶基因CET1的遗传分析将进行，从条件等位基因的分离和定点突变开始。突变体将被鉴定出对三磷酸酶活性和与鸟苷基转移酶亚基相互作用重要的区域。我们还将分析第二个酵母基因，该基因编码的蛋白质序列与封顶酶三磷酸酶相似。为了将capping的研究扩展到高等真核生物，我们克隆并分析了秀丽隐杆线虫的capping酶基因。该蛋白有一个区域与酵母鸟苷基转移酶同源，另一个区域与蛋白酪氨酸磷酸酶（PTP）家族相似。该结构域具有mRNA三磷酸酶活性，我们将确定是否使用ptp样机制。我们还将描述小鼠封顶酶，它可以在酵母中起作用。我们已经确定了几种ptp样蛋白与三磷酸酶有广泛的相似性，并将测试它们在RNA上的活性。mRNA capping是基因表达的一个基本过程。虽然许多方面在进化过程中是保守的，但酵母和高级真核生物的盖帽酶之间似乎存在重大差异，这些差异可能被用于抗真菌药物靶向。

mRNA undergoes several essential modifications, the first of which is addition of a 7-methylguanosine cap. Because capping occurs soon after transcription initiation and is specific to RNA polymerase II transcripts, we postulated that the capping and transcription machinery are physically or functionally associated. Using a combination of biochemical and genetic techniques, we showed that capping enzyme is recruited to the initiation complex by interacting specifically with the phosphorylated C-terminal domain (CTD) of RNA polymerase II. We will extend these observations to map interaction domains of the CTD and capping enzyme, determine which kinase(s) are responsible, how CTD binding affects capping activities, and at which point capping enzyme is released from the transcription complex. Yeast capping enzyme consists of two proteins: a triphosphatase and a guanylyltransferase. In contrast, higher eukaryotic capping enzymes consist of a single protein with both activities. A genetic analysis of the yeast triphosphatase gene CET1 will be carried out, beginning with isolation of conditional alleles and site-directed mutagenesis. The mutants will be characterized to identify regions important for triphosphatase activity and for interaction with the guanylyltransferase subunit. We will also analyze a second yeast gene that encodes a protein similar in sequence to the capping enzyme triphosphatase. To extend our studies of capping to higher eukaryotes, we cloned and analyzed the C. elegans capping enzyme gene. This protein has one region homologous to yeast guanylyltransferases and a second with similarity to the protein tyrosine phosphatase (PTP) family. This domain has mRNA triphosphatase activity and we will determine whether a PTP-like mechanism is used. We will also characterize the mouse capping enzyme, which can function in yeast. We have identified several PTP-like proteins with extensive similarity to the capping enzyme triphosphatase and will test them for activity on RNA. mRNA capping is a fundamental process in gene expression. Although many aspects are conserved over evolution, there appear to major differences between yeast and higher eukaryotic capping enzymes that could be exploited for antifungal drug targeting.