DIRECT MEASUREMENT OF THE POLIOVIRUS RNA-POLYMERASE ERROR FREQUENCY INVITRO
DIRECT MEASUREMENT OF THE POLIOVIRUS RNA-POLYMERASE ERROR FREQUENCY INVITRO
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DOI：
10.1128/jvi.62.2.558-562.1988
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发表时间：
1988-02-01
影响因子：
5.4
通讯作者：
FLANEGAN, JB
中科院分区：
文献类型：
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作者：
WARD, CD;STOKES, MAM;FLANEGAN, JB
通过在体外复制均聚RNA模板来检查脊髓灰质炎病毒RNA依赖性RNA聚合酶的RNA复制保真度。脊髓灰质炎病毒RNA聚合酶被广泛纯化，并用于复制具有等摩尔浓度的非互补和互补核糖核苷酸的poly（A）、poly（C）或poly（I）模板。错误频率表示为掺入的非互补核苷酸的量除以掺入的互补和非互补核苷酸的总量。聚合酶错误频率非常高，范围为7 × 10 - 4。10-4至5.4倍。10-3，取决于具体的反应条件。在给定的模板上用不同的非互补核苷酸底物获得的错误频率之间或在两个模板上确定的特定非互补底物的值之间没有显着差异。聚合酶对poly（U）和poly（G）的活性太低，无法测量这些模板上的错误频率。当反应条件从3.0 mM Mg 2+（pH 7.0）变为7.0 mM Mg 2+（pH 8.0）时，观察到错误频率增加了5倍。这种错误频率的增加与先前研究中在相同条件下观察到的伸长率增加八倍相关。
The fidelity of RNA replication by the poliovirus-RNA-dependent RNA polymerase was examined by copying homopolymeric RNA templates in vitro. The poliovirus RNA polymerase was extensively purified and used to copy poly(A), poly(C), or poly(I) templates with equimolar concentrations of noncomplementary and complementary ribonucleotides. The error frequency was expressed as the amount of a noncomplementary nucleotide incorporated divided by the total amount of complementary and noncomplementary nucleotide incorporated. The polymerase error frequencies were very high and ranged from 7 .times. 10-4 to 5.4 .times. 10-3, depending on the specific reaction conditions. There were no significant differences among the error frequencies obtained with different noncomplementary nucleotide substrates on a given template or between the values determined on two templates for a specific noncomplementary substrate. The activity of the polymerase on poly(U) and poly(G) was too low to measure error frequencies on these templates. A fivefold increase in the error frequency was observed when the reaction conditions were changes from 3.0 mM Mg2+ (pH 7.0) to 7.0 mM Mg2+ (pH 8.0). This increase in the error frequency correlates with an eightfold increase in the elongation rate that was observed under the same conditions in a previous study.