MRNA CAPPING ENZYME

MRNA经历了几个基本的修饰，第一个是添加了一个7-甲基鸟苷帽。由于封顶发生在转录启动后不久，并且是RNA聚合酶II转录本所特有的，我们假设封顶和转录机制在物理上或功能上是相关的。利用生化和遗传技术的结合，我们证明了封闭酶是通过与RNA聚合酶II的磷酸化的C末端结构域特异地相互作用而被招募到起始复合体中的。我们将把这些观察扩展到绘制CTD和封闭酶的相互作用结构域，确定哪个激酶(S)负责，CTD结合如何影响封顶活性，以及在什么时候封闭酶从转录复合体中释放。酵母封帽酶由两种蛋白质组成：三磷酸酶和鸟苷酸转移酶。相反，高等真核生物的封闭酶由一种同时具有两种活性的蛋白质组成。将对酵母三磷酸酶基因CET1进行遗传分析，首先分离条件等位基因和定点突变。将对突变体进行鉴定，以确定对三磷酸酶活性和与鸟苷酸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，它编码一种序列类似于封闭酶三磷酸酶的蛋白质。为了将盖帽的研究扩展到高等真核生物，我们克隆并分析了线虫盖帽酶基因。该蛋白有一个区域与酵母鸟苷转移酶同源，第二个区域与蛋白质酪氨酸磷酸酶(PTP)家族相似。这个结构域具有mRNA三磷酸酶活性，我们将确定是否使用了类似PTP的机制。我们还将对可以在酵母中发挥作用的小鼠封闭酶进行鉴定。我们已经确定了几个与封闭酶三磷酸酶有广泛相似性的PTP样蛋白，并将测试它们在RNA上的活性。信使核糖核酸封顶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，但酵母和更高级别的真核覆盖酶之间似乎存在重大差异，可以用于抗真菌药物的靶向。

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.