THE CCA-ADDING ENZYME (tRNA NUCLEOTIDYL TRANSFERASE)

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‘端CCA序列。与所有其他序列特异的RNA和DNA聚合酶不同，添加CCA的酶不使用核酸模板。因此，蛋白质本身必须以某种方式作为核苷酸加成的模板。虽然这两类不同的CCA添加酶只有一个保守的核苷酸转移酶基序，但这两类酶都有一个单一的活性位点，主要与tRNA的上半部分(“微螺旋”)结合，在CCA添加过程中不沿tRNA移位。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，我们提出了tRNA不断增长的3‘末端将逐渐压缩成一个口袋，允许单独的活性位点重复使用单个核苷酸结合位点。折叠的3‘末端如何决定CTP或ATP加成的特异性尚不清楚，但当挤压袋满时，核苷酸加成将停止。为了探索这一模型，我们现在提议对四种不同酶的活性部位、压缩口袋和tRNA结合区进行彻底的突变分析：古生菌I类柴胡CCA添加酶(Aim 1)，真细菌II类脂肪嗜热芽孢杆菌CCA添加酶(Aim 2)，以及Aquifex aeolicus罕见的II类CC和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元是单体还是多聚体(目标4)；我们将获得前四个目标中表征的选定突变体的晶体或共晶结构(目标5)；以及，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部位和皱缩袋，以创建具有改变序列特异性的突变体(目标6)。我们的实验应该揭示唯一使用蛋白质而不是核酸来模板特定核苷酸序列的酶的详细机制；揭示许多聚合酶用来促进在生长的3‘末端错误结合的核苷酸的启动和编辑的普遍性；并可能解释为什么今天由两个高度不同的蛋白质支架(I类和II类)执行这一古老的基本活动。

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).