THE CCA-ADDING ENZYME (tRNA NUCLEOTIDYL TRANSFERASE)

CCA 添加酶（tRNA 核苷酸转移酶）

• 批准号: 6769985
• 负责人: ALAN M WEINER
• 金额: $ 26.08万
• 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 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6769985
• 关键词: active sites; adenosine triphosphate; apoenzymes; bacterial proteins; binding sites; conformation; crosslink; crystallization; dimer; enzyme mechanism; enzyme model; enzyme structure; nucleic acid sequence; nucleotidyltransferase; protein structure function; transfer RNA

DESCRIPTION (provided by applicant): 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. 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 somehow serve as a template for nucleotide addition. Although the two nonhomologous classes of CCA adding enzymes share only a conserved nucleotidyltransferase motif, both classes have a single active site, bind primarily to the top half ("minihelix") of tRNA, and do not translocate along the tRNA during CCA addition. To explain how three nucleotides can be added without movement of the tRNA or active site, we proposed that the growing 3' terminus of the tRNA would progressively scrunch into a pocket, allowing the solitary active site to reuse a single nucleotide-binding site. How the folded 3' terminus would determine the specificity of CTP or ATP addition was not clear, but nucleotide addition would cease when the scrunching pocket was full. To explore this model, we now propose a thorough mutational analysis of the active site, scrunching pocket, and tRNA-binding regions of four different enzymes: the archaeal class I Sulfolobus shibatae CCA-adding enzyme (Aim 1), the eubacterial class II Bacillus stearothermophilus CCA-adding enzyme (Aim 2), and the unusual eubacterial class II CC- and A-adding enzymes of Aquifex aeolicus (Aim 3). In addition, we will mutate the dimerization interfaces of both class I and class II enzymes to determine whether the functional unit of these enzymes is a monomer or multimer (Aim 4); we will obtain crystal or cocrystal structures of selected mutants characterized in the previous four aims (Aim 5); and, as the ultimate test of our understanding, we will use structure-based protein redesign of the nucleotide binding site and scrunching pocket to create mutants with altered sequence specificity (Aim 6). Our experiments should reveal the detailed mechanism of the only enzyme that templates specific nucleotide sequences using protein instead of nucleic acid; shed light on the generality of the scrunching mechanisms used by many polymerases to facilitate initiation as well as editing of misincorporated nucleotides at the growing 3' terminus; and possibly explain why this ancient essential activity is performed today by two highly divergent protein scaffolds (class I and II).

描述（由申请人提供）： CCA添加酶[ATP（CTP）：tRNA核苷酸转移酶]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，蛋白质本身必须以某种方式用作添加核苷酸的模板。尽管两种非同源类CCA添加酶仅具有一个保守的核苷酸转移酶基序，但两个类别都有一个单个活性位点，主要与TRNA的上半部分（“ Minihelix”）结合，并且在CCA添加过程中不会沿TRNA转换。为了解释如何在不移动tRNA或活性位点的情况下添加三个核苷酸，我们提出，tRNA的生长3'末端将逐渐刮入口可袋，从而使孤立的活性位点可以重复使用单个核苷酸结合位点。折叠的3'末端将如何确定CTP或ATP添加的特异性尚不清楚，但是当经板口袋满时，核苷酸添加将停止。 To explore this model, we now propose a thorough mutational analysis of the active site, scrunching pocket, and tRNA-binding regions of four different enzymes: the archaeal class I Sulfolobus shibatae CCA-adding enzyme (Aim 1), the eubacterial class II Bacillus stearothermophilus CCA-adding enzyme (Aim 2), and the unusual eubacterial class II CC- and Aquifex aeolicus的A添加酶（AIM 3）。此外，我们将突变I类和II类酶的二聚化界面，以确定这些酶的功能单位是单体还是多聚体（AIM 4）；我们将获得前四个目标中特征的选定突变体的晶体或共晶结构（AIM 5）；并且，作为对我们理解的最终测试，我们将使用基于结构的蛋白质重新设计核苷酸结合位点和串行口袋来创建具有序列特异性改变的突变体（AIM 6）。我们的实验应揭示唯一使用蛋白质代替核酸来模拟特定核苷酸序列的唯一酶的详细机制。阐明了许多聚合酶用于促进启动以及在生长的3'末端促进构成核苷酸的一般性的一般性；并且可能解释了为什么今天有两个高度不同的蛋白质支架（I和II类）进行这种古老的基本活动。