MRNA CAPPING ENZYME

mRNA加帽酶

• 批准号: 2752351
• 负责人: Stephen Buratowski
• 金额: $ 18.88万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-01 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2752351
• 关键词: DNA directed RNA polymerase; acid anhydride hydrolase; allosteric site; chemical binding; enzyme activity; enzyme mechanism; enzyme structure; fungal genetics; gene interaction; genetic transcription; messenger RNA; nucleotidyltransferase; phosphorylation; posttranscriptional RNA processing; protein kinase; protein tyrosine phosphatase; site directed mutagenesis; species difference; transcription factor

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.

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。 由于加帽发生在转录起始后不久，并且是 RNA 聚合酶 II 转录本特异的，因此我们假设加帽和转录机制在物理或功能上相关。 通过结合生化和遗传技术，我们发现加帽酶通过与 RNA 聚合酶 II 的磷酸化 C 末端结构域 (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结果，以绘制 CTD 和加帽酶的相互作用域图，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时间点。酵母加帽酶由两种蛋白质组成：三磷酸酶和鸟苷酸转移酶。 相反，高等真核生物的加帽酶由具有两种活性的单一蛋白质组成。将从条件等位基因的分离和定点诱变开始，对酵母三磷酸酶基因 CET1 进行遗传分析。 对突变体进行表征，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，该基因编码与加帽酶三磷酸酶序列相似的蛋白质。为了将我们的加帽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加帽酶基因。 该蛋白质有一个区域与酵母鸟苷酸转移酶同源，第二个区域与蛋白质酪氨酸磷酸酶 (PTP) 家族相似。 该结构域具有 mRNA 三磷酸酶活性，我们将确定是否使用类似 PTP 的机制。 我们还将表征小鼠加盖酶，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加帽是基因表达的一个基本过程。尽管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，可用于抗真菌药物靶向。