CCA-ADDING ENZYME (TRNA NUCLEOTIDYLTRANSFERASE)

CCA添加酶[ATP（CTP）：tRNA核苷酸转移酶]使用CTP和ATP作为底物，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，蛋白质本身必须充当模板，或者酶必须使用新的机制来指定核苷酸添加。 我们已经表明，CCA添加酶只有一个单一的活性位点，该酶主要结合到tRNA的受体茎（“上半部分”），并在添加CCA的过程中，tRNA在酶表面保持不动。 为了解释三个核苷酸是如何被添加到tRNA上而不移动tRNA或活性位点的，我们提出了tRNA的3'末端逐渐折叠，以允许单独的活性位点重新使用单个核苷酸结合位点。 ATP结合位点将由重折叠的CC末端和酶协同产生，并且当核苷酸结合口袋充满时，核苷酸添加将停止。 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。 在这里，我们建议研究CCA添加酶的生物化学细节。 这些实验将测试协同模板模型，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法来识别活性位点、核苷酸结合口袋和tRNA结合位点附近的氨基酸残基;我们将询问这些残基中的突变是否会改变CCA添加的特异性，正如模型所预测的那样;我们将使用核苷酸类似物来定义核苷酸结合口袋的性质;我们将继续努力使添加CCA的酶与tRNA底物结晶或共结晶。 原则上，这种酶与三种底物（tRNA-N、tRNA-NC、tRNA-NCC）和成熟tRNA产物（tRNA-NCCA，其中N是“碱基”）的共晶体结构将提供这种不寻常的酶作用的动态图像。

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.