Stopped-flow fluorescence kinetic study of protein sliding and intersegment transfer in the target DNA search process.
Stopped-flow fluorescence kinetic study of protein sliding and intersegment transfer in the target DNA search process.
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DOI：
10.1016/j.jmb.2013.09.019
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发表时间：
2014-01-09
影响因子：
5.6
通讯作者：
Iwahara J
中科院分区：
文献类型：
--
作者：
Esadze A;Iwahara J
蛋白质在DNA上易位的动力学表征是非平凡的，因为同时存在多种不同的机制使得提取特定于特定易位机制的信息变得困难。在这项研究中，我们开发了新的方法，用于靶DNA搜索过程中蛋白质滑动和片段间转移（也称为“直接转移”）的动力学研究。基于离散状态随机模型平均搜索时间的解析表达式，我们导出了涉及竞争DNA和片段间转移机制的系统中蛋白质-靶标关联的表观速率常数kapp的解析形式。我们的kapp解析形式便于实验确定靶结合过程中节间转移和滑动的动力学速率常数。利用靶结合动力学的停流荧光数据以及kapp的分析形式，我们研究了Egr-1锌指蛋白在靶DNA结合过程中的易位。使用DNA长度依赖的kapp数据分析滑动。利用kapp对竞争对手DNA浓度的依赖性，我们确定了片段间转移的二阶速率常数。我们的研究结果表明，Egr-1锌指蛋白的靶结合过程中的一个主要途径是涉及到片段间转移到非特异性位点并随后滑动到靶标。
Kinetic characterizations of protein translocation on DNA are nontrivial because the simultaneous presence of multiple different mechanisms makes it difficult to extract the information specific to a particular translocation mechanism. In this study, we have developed new approaches for the kinetic investigations of proteins’ sliding and intersegment transfer (also known as ‘direct transfer’) in the target DNA search process. Based on the analytical expression of the mean search time for the discrete–state stochastic model, we derived analytical forms of the apparent rate constant kapp for protein-target association in systems involving competitor DNA and the intersegment transfer mechanism. Our analytical forms of kapp facilitate the experimental determination of the kinetic rate constants for intersegment transfer and sliding in the target association process. Using stopped-flow fluorescence data for the target association kinetics along with the analytical forms of kapp, we have studied the translocation of the Egr-1 zinc-finger protein in the target DNA association process. Sliding was analyzed using the DNA length-dependent kapp data. Using the dependence of kapp on the concentration of competitor DNA, we determined the second-order rate constant for intersegment transfer. Our results indicate that a major pathway in the target association process for the Egr-1 zinc-finger protein is the one involving intersegment transfer to a nonspecific site and the subsequent sliding to the target.
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影响因子：
16.8
作者：
通讯作者：
--
影响因子：
5.6
作者：
BRESLOFF, JL;CROTHERS, DM
通讯作者：
CROTHERS, DM
DOI：
10.1073/pnas.0805050105
发表时间：
2008-09-16
影响因子：
11.1
作者：
Doucleff, Michaeleen;Clore, G. Marius
通讯作者：
Clore, G. Marius
DOI：
10.1016/s0378-4371(02)01038-5
发表时间：
2002-10-01
影响因子：
3.3
作者：
Bruinsma, RF
通讯作者：
Bruinsma, RF
影响因子：
3.6
作者：
Iwahara, Junji;Levy, Yaakov
通讯作者：
Levy, Yaakov