MRNA CAPPING ENZYME

mRNA加帽酶

• 批准号: 6180625
• 负责人: Stewart H Shuman
• 金额: $ 35.05万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-05-01 至 2003-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6180625
• 关键词: DNA directed RNA polymerase; Saccharomyces cerevisiae; X ray crystallography; acid anhydride hydrolase; active sites; crystallization; enzyme complex; enzyme mechanism; enzyme structure; fungal genetics; fungal proteins; gene expression; messenger RNA; nucleotidyltransferase; posttranscriptional RNA processing; precursor mRNA; protein protein interaction; site directed mutagenesis

mRNA processing plays an important role in the regulated expression of eukaryotic genes. Processing occurs contranscriptionally as nascent chains are being synthesized by nuclear RNA polymerase II. The earliest modification event is the formation of the m7GpppN cap. Our goal is to understand the mechanism of capping and the role f the cap in cellular mRNA metabolism through biochemical and genetic analysis of enzymes that catalyze cap formation. This proposal focuses on the first two enzymes in the cap synthetic pathway: RNA triphosphatase and RNA guanlyltransferase. Yeast and mammals use different strategies to assemble a bifunctional enzyme with triphosphatase and guaylyltransferase activities. In yeast, separate triphosphatase (Cet1p) and guaylyltransferase (Ceg1p) enzymes interact to form a heterodimer, whereas in mammals, autonomous triphosphatase and guaylyltransferase domains are link in cis within a single polypeptide (Mce1p). The guanylytransferases are conserved between fungi and mammals, but the triphosphatase components diverge with respect to structure and mechanism. We propose in aim 1 to define the active sites of the mouse and yeast triphosphatases by targeted mutagenesis and to crystallize the enzymes for structure determination by X-ray diffraction. In aim 2, we will explore how the capping apparatus is targeted in vivo to achieve specific capping of RNA polymerase II transcripts. Our working model is that capping is directed to nascent pre-mRNAs though the binding of the guanylyltransferase component to the phosphorylated CTD of elongating RNA polymerase II. We will define the CTD-guanylyltransferase interface and test the hypothesis that the triphosphatase is brought to the transcription complex via its physical connection to the guanylyltranserase. In aim 3, we propose to define the features of yeast triphosphatase and guanylyltranserfase that mediate heterodimerization. In aim 4, we will exploit a collection of temperature-sensitive cet1 mutants to determine how inactivation of RNA triphosphatase affects gene expression. The studies of the yeast and mammalian capping enzymes proposed herein will provide new insights into phosphoryl transfer reaction mechanisms, contribute to an emerging picture of the pol II CTD as a landing pad for macromolecular assemblies that regulate mRNA synthesis and processing, and help clarify the role of 5' end structure in mRNA decay.

mRNA处理在调节表达中起重要作用 真核基因。 处理在 核RNA聚合酶II合成新生链。 最早的修改事件是M7GPPPN盖的形成。 我们的目标是了解上限机制和作用 细胞mRNA代谢中的帽通过生化和遗传 分析催化帽形成的酶。 这个建议 重点关注盖合成途径中的前两个酶：RNA 三磷酸酶和RNA鸟酰转移酶。 酵母和哺乳动物使用 不同的策略来组装双功能酶 三磷酸酶和瓜伊化罗转移酶活性。 在酵母中，分开 三磷酸酶（CET1P）和瓜伊lyltransferase（CEG1P）酶 相互作用以形成异二聚体，而在哺乳动物中，自主 三磷酸酶和瓜伊llyltransferase结构域是顺式中的链接 单个多肽（MCE1P）。 Guanylylytransferases是保存的 在真菌和哺乳动物之间，但三磷酸酶成分发散 关于结构和机制。 我们建议AIM 1 定义小鼠和酵母三磷酸酶的活性位点 靶向诱变并结晶酶以进行结构 通过X射线衍射测定。 在AIM 2中，我们将探讨如何 上限设备的靶向体内以实现特定的上限 RNA聚合酶II转录物的。 我们的工作模型是上限 通过结合，针对新生的前mrNA Guanylylltransferase成分到延长的磷酸化CTD RNA聚合酶II。 我们将定义CTD-Guanyllyltransferase酶 界面并检验带有三磷酸酶的假设 通过其物理连接到转录复合物 Guanylyltranserase。 在AIM 3中，我们建议定义 酵母三磷酸酶和Guanylyltranserfase介导 异二聚体。 在AIM 4中，我们将利用 温度敏感的CET1突变体以确定如何失活 RNA三磷酸酶影响基因表达。 酵母的研究 本文提出的哺乳动物上限酶将提供新的 对磷酸化转移反应机制的见解，有助于 Pol II CTD的新兴图片作为着陆垫 调节mRNA合成和的大分子组件 处理，并有助于阐明5'端结构在mRNA中的作用 衰变。