Determination of relative rate constants for in vitro RNA processing reactions by internal competition.
Determination of relative rate constants for in vitro RNA processing reactions by internal competition.
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DOI：
10.1016/j.ab.2014.08.022
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发表时间：
2014-12-15
影响因子：
2.9
通讯作者：
Harris ME
中科院分区：
文献类型：
--
作者：
Lin HC;Yandek LE;Gjermeni I;Harris ME
RNA识别和催化的研究通常涉及通过将底物或产物浓度的变化拟合为指数或线性函数来测量单个RNA序列变异反应的速率常数。一种互补的方法是通过内部竞争来确定相对速率常数，这涉及到在含有多种底物的反应中，对底物或产物比率的随时间变化进行量化。在这里，我们回顾了通过分析底物和产物比率来确定相对速率常数的方法，并以核糖核酸酶P对前体tRNA的体外处理为模型系统说明了它们的应用。在涉及RNA底物的反应分析中，无活性底物群体的存在是一个常见的复杂因素，并说明了对观察到的速率常数进行定量校正的方法。这些结果以及最近在文献中的应用表明，内部竞争为使用标准分子生物学方法分析RNA加工动力学提供了一种替代方法，可以直接量化底物特异性，并可能扩展到一系列应用。
Studies of RNA recognition and catalysis typically involve measurement of rate constants for reactions of individual RNA sequence variants by fitting changes in substrate or product concentration to exponential or linear functions. A complementary approach is determination of relative rate constants by internal competition, which involves quantifying the time dependent changes in substrate or product ratios in reactions containing multiple substrates. Here, we review approaches for determining of relative rate constants by analysis of both substrate and product ratios and illustrate their application using the in vitro processing of precursor tRNA by ribonuclease P as a model system. The presence of inactive substrate populations is a common complicating factor in analysis of reactions involving RNA substrates, and approaches for quantitative correction of observed rate constants for these effects are illustrated. These results together with recent applications in the literature indicate that internal competition offers an alternate method for analyzing RNA processing kinetics using standard molecular biology methods that directly quantifies substrate specificity and may be extended to a range of applications.
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影响因子：
15
作者：
Harris, Michael E.;Dai, Qing;Anderson, Vernon E.
通讯作者：
Anderson, Vernon E.
影响因子：
2.9
作者：
Bertram JG;Oertell K;Petruska J;Goodman MF
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Strobel, Scott A.
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通讯作者：
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14.8
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