CCA-ADDING ENZYME (TRNA NUCLEOTIDYLTRANSFERASE)

CCA 添加酶（TRNA 核苷酸转移酶）

• 批准号: 6339995
• 负责人: ALAN M WEINER
• 金额: $ 19.33万
• 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/6339995
• 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核苷酸基转移酶]以CTP和ATP为底物，每次添加一个核苷酸，构建并修复所有tRNA的3'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，蛋白质本身必须充当模板，或者酶必须使用一种新的机制来指定核苷酸的添加。我们已经证明，添加CCA的酶只有一个活性位点，该酶主要与tRNA的受体茎（“上半部分”）结合，并且在添加CCA期间，tRNA在酶表面保持不动。为了解释三个核苷酸是如何在不移动tRNA或活性位点的情况下被添加到tRNA上的，我们提出tRNA生长的3'端逐渐重新折叠，以允许单独的活性位点重复使用单个核苷酸结合位点。ATP结合位点将由重新折叠的CC末端和酶共同产生，当核苷酸结合袋满时，核苷酸的添加将停止。因此，添加CCA的模板将是一个动态的核糖核蛋白结构，我们称之为协同模板。本文拟对cca添加酶进行生化细节研究。实验将验证协作模板模型，并提供有关cca添加酶的丰富新信息。具体来说，我们将使用光化学交联和羟基自由基足迹来识别活性位点、核苷酸结合袋和tRNA结合位点附近的氨基酸残基；我们将询问这些残基的突变是否会像模型预测的那样改变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；我们将继续努力用tRNA底物结晶或共结晶CCA添加酶。原则上，这种酶与三种底物（tRNA-N、tRNA-NC、tRNA- ncc）和成熟的tRNA产物（tRNA- ncca，其中N是“鉴别碱基”）的共晶结构将提供这种不寻常的酶在作用中的动态图像。