Pre-steady-state kinetics of initiation of transcription by T7 RNA polymerase: a new kinetic model.

Pre-steady-state kinetics of initiation of transcription by T7 RNA polymerase: a new kinetic model.

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. In 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.

为了开始理解在模式噬菌体T7 RNA聚合酶系统中转录起始的机制，人们通过淬灭流动动力学对最简单的反应——二核苷酸的合成进行了研究，并利用特定机制的速率方程的数值积分来测试特定的动力学模型。为了拟合在预稳态动力学中观察到的时间依赖性，提出了一个二核苷酸合成模型，其中必须包括二核苷酸与酶 - DNA复合物的再结合。使用二核苷酸作为底物的单独反应证实了这一机制以及所确定的速率常数。在这些条件下观察到的作为抑制作用的二核苷酸再结合在较长转录本的合成中形成了一个有生成能力的中间产物，并且在未来的动力学机制中必须予以考虑。导致产物形成的限速步骤显示出与两个底物GTP分子的结合相一致的底物依赖性，并且在GTP饱和水平下，其幅度与产物释放速率相当。产物释放速率与GTP浓度呈正相关，这表明该反应显示出碱基特异性的底物激活。另一个底物分子的结合（大概是通过与三磷酸结合位点的相互作用）可能促进二核苷酸产物从复合物中置换出来。