MRNA CAPPING ENZYME

mRNA经历了几个重要的修饰，其中第一个是添加7-甲基鸟苷帽。 由于加帽发生后不久，转录起始，是特定的RNA聚合酶II转录，我们假设，加帽和转录机制的物理或功能相关。 使用生物化学和遗传技术的组合，我们表明，加帽酶被招募到起始复合物的相互作用，特异性与磷酸化的C-末端结构域（CTD）的RNA聚合酶II。 我们将扩展这些观察，以映射CTD和加帽酶的相互作用域，确定哪些激酶负责，CTD结合如何影响加帽活性，以及加帽酶从转录复合物中释放的位置。酵母加帽酶由两种蛋白质组成：三磷酸酶和鸟苷酰转移酶。 相比之下，高等真核生物的加帽酶由具有两种活性的单一蛋白质组成。将进行酵母三磷酸酶基因CET 1的遗传分析，从条件等位基因的分离和定点诱变开始。 突变体将被表征以鉴定对三磷酸酶活性和与鸟苷酰转移酶亚基的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加帽酶三磷酸酶相似。为了将我们的研究扩展到高等真核生物，我们克隆并分析了C。elegans加帽酶基因 该蛋白质具有一个与酵母鸟苷酰转移酶同源的区域和第二个与蛋白酪氨酸磷酸酶（PTP）家族相似的区域。 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。 我们还将表征小鼠加帽酶，它可以在酵母中发挥作用。 我们已经鉴定出几种与加帽酶三磷酸酶具有广泛相似性的PTP样蛋白，并将测试它们对RNA的活性。mRNA加帽是基因表达的基本过程。虽然许多方面在进化过程中是保守的，但酵母和高等真核生物加帽酶之间似乎存在重大差异，这些差异可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.