Assembly, purification, and pre-steady-state kinetic analysis of active RNA-dependent RNA polymerase elongation complex.
Assembly, purification, and pre-steady-state kinetic analysis of active RNA-dependent RNA polymerase elongation complex.
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DOI：
10.1074/jbc.m111.325530
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发表时间：
2012-03-23
期刊：
影响因子：
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通讯作者：
Klumpp K
中科院分区：
文献类型：
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作者：
Jin Z;Leveque V;Ma H;Johnson KA;Klumpp K
背景：以往的研究未能重建丙型肝炎病毒（HCV） RNA依赖性RNA聚合酶的活性复制复合体。结果：完成了HCV复制复合体的组装、纯化和鉴定。结论：HCV聚合酶可形成高活性的复制复合体，催化RNA的快速复制。意义：纯化的活性复制复合体对于机制研究和药物发现至关重要。NS5B是一种RNA依赖的RNA聚合酶，负责复制丙型肝炎病毒（HCV）基因组RNA。尽管经过十多年的研究，高活性NS5B聚合酶·RNA复合物的形成机制和结构研究仍然难以捉摸。在这里，我们报告说，通过一种优化起始条件的新方法，我们能够产生一个高产的NS5B·引物·模板延伸复合物，在9核苷酸引物形成后停滞。与之前报道的活性NS5B的比例非常低相反，我们观察到在优化条件下，高达65%的NS5B可以转化为活性延伸配合物。延伸络合物非常稳定，允许纯化远离多余的核苷酸和流产起始产物，因此纯化的络合物适用于聚合酶活性的预稳态动力学分析。单次转化动力学研究表明，CTP与表观Kd和kpol分别为39±3 μm和16±1 s−1，kpol/Kd的特异性常数为0.41 μm−1 s−1。同时测定了多核苷酸结合的动力学过程。这项工作建立了一种新的方法来产生具有重要医学意义的NS5B聚合酶的高活性延伸复合物，用于结构和功能研究。
Background: Previous studies have failed to reconstitute an active replication complex with hepatitis C virus (HCV) RNA-dependent RNA polymerase. Results: The replication complex from HCV was assembled, purified, and characterized. Conclusion: A highly active replication complex can be formed with HCV polymerase that catalyzes fast and processive RNA replication. Significance: A purified and active replication complex is essential for mechanistic studies and drug discovery. NS5B is the RNA-dependent RNA polymerase responsible for replicating hepatitis C virus (HCV) genomic RNA. Despite more than a decade of work, the formation of a highly active NS5B polymerase·RNA complex suitable for mechanistic and structural studies has remained elusive. Here, we report that through a novel way of optimizing initiation conditions, we were able to generate a productive NS5B·primer·template elongation complex stalled after formation of a 9-nucleotide primer. In contrast to previous reports of very low proportions of active NS5B, we observed that under optimized conditions up to 65% of NS5B could be converted into active elongation complexes. The elongation complex was extremely stable, allowing purification away from excess nucleotide and abortive initiation products so that the purified complex was suitable for pre-steady-state kinetic analyses of polymerase activity. Single turnover kinetic studies showed that CTP is incorporated with apparent Kd and kpol values of 39 ± 3 μm and 16 ± 1 s−1, respectively, giving a specificity constant of kpol/Kd of 0.41 μm−1 s−1. The kinetics of multiple nucleotide incorporation during processive elongation also were determined. This work establishes a novel way to generate a highly active elongation complex of the medically important NS5B polymerase for structural and functional studies.