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首页> 外文期刊>Journal of Molecular Biology >ATP-dependent Translocation of Proteins along Single-stranded DNA: Models and Methods of Analysis of Pre-steady State Kinetics.
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ATP-dependent Translocation of Proteins along Single-stranded DNA: Models and Methods of Analysis of Pre-steady State Kinetics.

机译：沿着单链DNA的ATP依赖性蛋白质转运：稳态前动力学分析的模型和方法。

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摘要

Processive DNA helicases are able to translocate along single-stranded DNA (ssDNA) with biased directionality in a nucleoside triphosphate-dependent reaction, although translocation is not generally sufficient for helicase activity. An understanding of the mechanism of protein translocation along ssDNA requires pre-steady state transient kinetic experiments. Although ensemble experimental approaches have been developed recently for the study of translocation of proteins along DNA, quantitative analysis of the complete time-courses from these experiments, which is needed to obtain quantitative estimates of translocation kinetic parameters (rate constants, processivity, step sizes and ATP coupling) has been lacking. We discuss three ensemble transient kinetic experiments that can be used to study protein translocation along ssDNA, along with the advantages and limitations of each approach. We further describe methods to analyze the complete kinetic time-courses obtained from such experiments performed with a series of ssDNA lengths under "single-round" conditions (i.e. in the absence of re-binding of dissociated protein to DNA). These analysis methods utilize a sequential "n-step" model for protein translocation along ssDNA and enable quantitative determinations of the rate constant, processivity and step size for translocation through global non-linear least-squares fitting of the full time-courses.
机译：进行性DNA解旋酶能够在核苷三磷酸依赖性反应中以方向性偏向的方式沿单链DNA（ssDNA）进行移位，尽管通常移位对于解旋酶活性而言并不足够。对蛋白质沿ssDNA转运的机制的了解需要进行稳态前瞬态动力学实验。尽管最近已经开发了整体实验方法来研究蛋白质沿DN​​A的易位性，但是从这些实验中对完整时间过程进行定量分析是获得易位动力学参数（速率常数，持续性，步长和ATP耦合）一直缺乏。我们讨论了三个整体瞬态动力学实验，可用于研究沿着ssDNA的蛋白质转运，以及每种方法的优点和局限性。我们进一步描述了分析从这样的实验中获得的完整动力学时程的方法，这些实验是在“单轮”条件下（即在不存在解离蛋白与DNA的重新结合的情况下）用一系列ssDNA长度进行的。这些分析方法利用顺序的“ n步”模型沿ssDNA进行蛋白质移位，并通过全时程的全局非线性最小二乘拟合对移位的速率常数，持续性和步长进行定量测定。

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