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首页> 外文期刊>Journal of Molecular Biology >Pre-steady-state kinetics of initiation of transcription by T7 RNApolymerase: A new kinetic model
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Pre-steady-state kinetics of initiation of transcription by T7 RNApolymerase: A new kinetic model

机译：T7 RNA聚合酶引发转录的稳态前动力学：一种新的动力学模型

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摘要

In order to begin to understand the mechanism of the initiation of transcription in the model bacteriophage T7 RNA polymerase system, the simplest possible reaction, the synthesis of a dinucleotide, has been followed by quench-flow kinetics and numerical integration of mechanism specific rate equations has been used to test specific kinetic models. Zn order to fit the observed time dependence in the pre-steady-state kinetics, a model for dinucleotide synthesis is proposed in which rebinding of the dinucleotide to the enzyme-DNA complex must be included. Separate reactions using dinucleotide as a substrate confirm this mechanism and the determined rate constants. The dinucleotide rebinding observed as inhibition under these conditions forms a productive intermediate in the synthesis of longer transcripts, and must be included in future kinetic mechanisms. The rate-limiting step leading to product formation shows a substrate dependence consistent with the binding of two substrate GTP molecules, and at saturating levels of GTP, is comparable in magnitude to the product release rate. The rate of product release shows a positive correlation with the concentration of GTP, suggesting that the reaction shows base-specific substrate activation. The binding of another substrate molecule, presumably via interaction with the triphosphate binding site, likely facilitates displacement of the dinucleotide product from the complex.
机译：为了开始理解模型噬菌体T7 RNA聚合酶系统中转录起始的机制，最简单的反应，即二核苷酸的合成，随后是猝灭流动动力学和机制特定速率方程的数值积分。用于测试特定的动力学模型。为了使Zn适合于稳态前动力学中观察到的时间依赖性，提出了一种用于二核苷酸合成的模型，其中必须包括二核苷酸与酶-DNA复合物的重新结合。使用二核苷酸作为底物的单独反应证实了该机理和确定的速率常数。在这些条件下被抑制的二核苷酸重新结合形成了较长转录本合成中的生产性中间体，并且必须包括在未来的动力学机制中。导致产物形成的限速步骤显示出与两个底物GTP分子的结合一致的底物依赖性，并且在GTP的饱和水平下，其量级与产​​物释放速率相当。产物释放的速率与GTP浓度呈正相关，表明该反应具有碱基特异性底物活化作用。大概通过与三磷酸结合位点的相互作用，另一种底物分子的结合可能促进二核苷酸产物从复合物中的置换。

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