CCA-ADDING ENZYME (TRNA NUCLEOTIDYLTRANSFERASE)

CCA 添加酶（TRNA 核苷酸转移酶）

• 批准号: 6386588
• 负责人: ALAN M WEINER
• 金额: $ 21.59万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-01 至 2003-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6386588
• 关键词: active sites; adenosine triphosphate; binding sites; crosslink; crystallization; cytosine nucleotides; enzyme mechanism; enzyme model; enzyme structure; enzyme substrate; enzyme substrate analog; enzyme substrate complex; nucleic acid sequence; nucleotide analog; nucleotidyltransferase; transfer RNA

The CCA-adding enzyme [ATP(CTP):tRNA nucleotidyltransferase] builds and repairs the 3' terminal CCA sequence of all tRNAs by adding one nucleotide at a time, using CTP and ATP as substrates. Unlike all other sequence-specific RNA and DNA polymerases, the CCA-adding enzyme does not use a nucleic acid template. Thus the protein itself must serve as a template, or the enzyme must use a novel mechanism to specify nucleotide addition. We have shown that the CCA-adding enzyme has only a single active site, that the enzyme binds primarily to the acceptor stem ("top half") of tRNA, and that the tRNA remains immobile on the enzyme surface during addition of CCA. To explain how three nucleotides can be added to tRNA without movement of either the tRNA or the active site, we proposed that the growing 3' terminus of the tRNA progressively refolds to allow the solitary active site to reuse a single nucleotide binding site. The ATP binding site would be created collaboratively by the refolded CC terminus and the enzyme, and nucleotide addition would cease when the nucleotide binding pocket is full. The template for CCA addition would therefore be a dynamic ribonucleoprotein structure, in a mechanism we call collaborative templating. Here we propose to study the CCA-adding enzyme in biochemical detail. The experiments will test the collaborative templating model, and provide a wealth of new information about the CCA-adding enzyme. Specifically, we will use photochemical crosslinking and hydroxyl radical footprinting to identify amino acid residues in the immediate vicinity of the active site, the nucleotide binding pocket, and the tRNA binding site; we will ask whether mutations in these residues change the specificity of CCA addition as predicted by the model; we will use nucleotide analogues to define the nature of the nucleotide binding pocket; and we will continue our efforts to crystallize or cocrystallize the CCA- adding enzyme with tRNA substrates. In principle, cocrystal structures of the enzyme with the three substrates (tRNA-N, tRNA- NC, tRNA-NCC) and the mature tRNA product (tRNA-NCCA where N is the "discriminator base") would provide a moving picture of this unusual enzyme in action.

CCA添加酶[ATP（CTP）：TRNA核苷酸转移酶]通过使用CTP和ATP作为底物添加一个核苷酸，通过一次添加一个核苷酸来构建和修复所有TRNA的3'末端CCA序列。与所有其他序列特异性RNA和DNA聚合酶不同，CCA添加酶不使用核酸模板。 因此，蛋白质本身必须用作模板，否则酶必须使用新颖的机制来指定核苷酸的添加。 我们已经表明，CCA添加酶只有一个活性位点，该酶主要与受体茎（“上半”）结合，并且在添加CCA期间，tRNA在酶表面上保持不动。 为了解释如何在不移动tRNA或活性位点的情况下将三个核苷酸添加到tRNA中，我们提出，tRNA的生长3'末端逐渐重新折叠以允许孤立的活性位点重复使用单个核苷酸结合位点。 ATP结合位点将由重折叠的CC末端和酶协作创建，而当核苷酸结合口袋满足时，核苷酸的添加将停止。 因此，CCA添加的模板将是一种动态的核糖核蛋白结构，我们称为协作模板。 在这里，我们建议通过生化细节研究CCA添加酶。 该实验将测试协作模型模型，并提供有关CCA添加酶的大量新信息。具体而言，我们将使用光化学交联和羟基自由基足迹来鉴定活性位点附近，核苷酸结合口袋和tRNA结合位点的氨基酸残基；我们将询问这些残基中的突变是否会如模型所预测的那样改变CCA添加的特异性。我们将使用核苷酸类似物来定义核苷酸结合袋的性质。我们将继续我们的努力来结晶或结合用tRNA底物添加CCA-添加酶。 原则上，酶与三个底物（TRNA-N，TRNA-NC，TRNA-NCC）和成熟的TRNA产物（trna-nCCA，n是“歧视基础”）的共晶结构将提供这种不寻常酶在作用中的动态图片。