Poliovirus RNA-dependent RNA polymerase (3Dpol) -: Assembly of stable, elongation-competent complexes by using a symmetrical primer-template substrate (sym/sub)
Poliovirus RNA-dependent RNA polymerase (3Dpol) -: Assembly of stable, elongation-competent complexes by using a symmetrical primer-template substrate (sym/sub)
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DOI：
10.1074/jbc.275.8.5329
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发表时间：
2000-02-25
影响因子：
4.8
通讯作者：
Cameron, CE
中科院分区：
文献类型：
--
作者：
Arnold, JJ;Cameron, CE
脊髓灰质炎病毒依赖RNA的RNA聚合酶3D(Pol1)催化的核苷酸掺入动力学和机理的详细研究受到限制，因为无法组装允许通过延伸末端标记的引物来监测活性的延伸复合体。我们已经解决了这个问题，采用了一种短的、对称的、杂多聚体的RNA引物模板，我们称之为“sym/sub.”3D(POL)-SYM/SUB络合物的形成较慢，这是因为3D(POL)-SYM/SUB与SYM/SUB的缔合速度较慢(0.1muM~(-1)S(-1))，以及3D(POL)-SYM/SUB络合物的异构化速度较慢(0.076·S(-1))，这是该络合物具有催化活性的先决条件。在消除竞争反应(如酶失活)的条件下，复杂的组装是化学计量的。在缺乏核苷酸的反应缓冲液中，3D(Ol)在22℃时的最大失活速率为0.051 S(-1)。在此温度下，三磷酸腺苷保护3D(Pol)不被失活，K-0.5为37 mU M。一旦形成3D(Pol)-sym/Sub延伸复合体是稳定的(t(1/2)=22℃下2 h)，并且似乎只含有单一的聚合酶单体，在Mg2+存在下，AMP、2‘-DAMP和3’-DAMP分别以72、0.6和1 S(-1)的速率以3D(Pol)结合到SYM/SUB中。加入AMP后，3D(Ol)-sym/subproduct络合物在22℃下的半衰期为8h，其稳定性依赖于温度。在30摄氏度时，络合物的稳定性下降了2-8倍。复杂解离是引物利用的限速步骤。3D(POL)从模板末端解离的速度比从内部位置解离的速度快10倍。SYM/SUB系统将促进3D(Ol)的机械分析，并允许将依赖于RNA的RNA聚合酶与其他类型的核酸聚合酶进行直接的动力学和热力学比较。
Detailed studies of the kinetics and mechanism of nucleotide incorporation catalyzed by the RNA-dependent RNA polymerase from poliovirus, 3D(pol), have been Limited by the inability to assemble elongation complexes that permit activity to be monitored by extension of end-labeled primers. We have solved this problem by employing a short, symmetrical, heteropolymeric RNA primer-template that we refer to as "sym/sub." Formation of 3D(pol)-sym/sub complexes is slow owing to a slow rate of association (0.1 mu M-1 s(-1)) of 3D(pol) and sym/sub and a slow isomerization (0.076 s(-1)) of the 3D(pol)-sym/sub complex that is a prerequisite for catalytic competence of this complex. Complex assembly is stoichiometric under conditions in which competing reactions, such as enzyme inactivation, are eliminated. Inactivation of 3D(pol) occurs at a maximal rate of 0.051 s(-1) at 22 degrees C in reaction buffer lacking nucleotide. At this temperature, ATP protects 3D(pol) against inactivation with a K-0.5 of 37 mu M. Once formed, 3D(pol)-sym/sub elongation complexes are stable (t(1/2) = 2 h at 22 degrees C) and appear to contain only a single polymerase monomer, In the presence of Mg2+, AMP, 2'-dAMP, and 3'-dAMP are incorporated into sym/sub by 3D(pol) at rates of 72, 0.6, and 1 s(-1), respectively. After incorporation of AMP, 3D(pol)-sym/sub product complexes have a half-life of 8 h at 22 degrees C. The stability of 3D(pol)-sym/sub complexes is temperature-dependent. At 30 degrees C, there is a 2-8-fold decrease in complex stability. Complex dissociation is the rate-limiting step for primer utilization. 3D(pol) dissociates from the end of template at a rate 10-fold faster than from internal positions. The sym/sub system will facilitate mechanistic analysis of 3D(pol) and permit a direct kinetic and thermodynamic comparison of the RNA-dependent RNA polymerase to the other classes of nucleic acid polymerases.