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]通过每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充当核苷酸添加的模板。尽管两种非同源的CCA添加酶仅共享保守的核苷酸基转移酶基序，但两种酶都具有单一活性位点，主要结合tRNA的上半部分（“微螺旋”），并且在CCA添加期间不沿沿着tRNA移位。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的，我们提出，不断增长的tRNA的3'末端将逐渐紧缩成一个口袋，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添加的特异性尚不清楚，但当紧缩口袋充满时，核苷酸添加将停止。为了探索这个模型，我们现在提出了一个彻底的突变分析的活性位点，scrunching口袋，和tRNA结合区的四种不同的酶：古细菌I类柴田硫化叶菌CCA添加酶（目的1），真细菌II类嗜热脂肪芽孢杆菌CCA添加酶（目的2），和不寻常的真细菌II类CC-和A-添加酶的Aquifex aeolicus（目的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）;我们将获得在前四个目标中表征的所选突变体的晶体或共晶体结构（目标5）;作为对我们理解力的终极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挤压口袋，以产生具有改变的序列特异性的突变体（目标6）。我们的实验应该揭示唯一一种使用蛋白质而不是核酸模板化特定核苷酸序列的酶的详细机制;阐明许多聚合酶用于促进在生长的3'末端处错误掺入的核苷酸的起始以及编辑的挤压机制的普遍性;并可能解释为什么这种古老的基本活动今天由两种高度不同的蛋白质支架（I类和II类）执行。

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).