Single-molecule imaging reveals mechanisms of protein disruption by a DNA translocase.
Single-molecule imaging reveals mechanisms of protein disruption by a DNA translocase.
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作者：
在生理环境中，核酸移位酶必须作用于被其他蛋白质占据的底物，并且移位酶的越来越受重视的作用是催化蛋白质从RNA和DNA的置换。然而，关于必然发生的不可避免的碰撞知之甚少，蛋白质障碍物的命运以及它们从DNA中被驱逐的机制仍然未被探索。在这里，我们试图建立从DNA使用RecBCD作为模型系统的蛋白质置换的机制基础。使用纳米制造的DNA和多色单分子显微镜，我们可视化模型易位酶和不同的DNA结合蛋白质之间的碰撞在真实的时间。我们表明，DNA移位酶RecBCD可以破坏核心RNA聚合酶（RNAP），全酶，停滞的延伸复合物，并在头对头或头对尾方向转录RNAP，以及EcoRIE 111 Q，乳糖阻遏物，甚至核小体。RecBCD在碰撞过程中不会暂停，并且经常在将蛋白质从DNA中驱逐出来之前将其推入数千个碱基对。我们的结论是，RecBCD通过直接转导的化学机械力，而不需要特定的蛋白质-蛋白质相互作用的障碍，蛋白质可以从DNA中去除通过主动破坏机制，作用于过渡态中间体，因为它们被推从一个非特异性位点到下一个。
In physiological settings nucleic acid translocases must act on substrates occupied by other proteins, and an increasingly appreciated role of translocases is to catalyze protein displacement from RNA and DNA. However, little is known regarding the inevitable collisions that must occur, and the fate of protein obstacles and the mechanisms by which they are evicted from DNA remain unexplored. Here we sought to establish the mechanistic basis for protein displacement from DNA using RecBCD as a model system. Using nanofabricated curtains of DNA and multi-color single-molecule microscopy, we visualized collisions between a model translocase and different DNA-bound proteins in real time. We show that the DNA translocase RecBCD can disrupt core RNA polymerase (RNAP), holoenzyme, stalled elongation complexes, and transcribing RNAP in either head-to-head or head-to-tail orientations, as well as EcoRIE111Q, lac repressor and even nucleosomes. RecBCD did not pause during collisions and often pushed proteins thousands of base-pairs before evicting them from DNA. We conclude that RecBCD overwhelms obstacles through direct transduction of chemomechanical force with no need for specific protein-protein interactions, and that proteins can be removed from DNA through active disruption mechanisms that act on a transition state intermediate as they are pushed from one nonspecific site to the next.
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影响因子：
56.9
作者：
Elf, Johan;Li, Gene-Wei;Xie, X. Sunney
通讯作者：
Xie, X. Sunney
影响因子：
16
作者：
Guy, Colin P.;Atkinson, John;Gupta, Milind K.;Mahdi, Akeel A.;Gwynn, Emma J.;Rudolph, Christian J.;Moon, Peter B.;van Knippenberg, Ingeborg C.;Cadman, Chris J.;Dillingham, Mark S.;Lloyd, Robert G.;McGlynn, Peter
通讯作者：
McGlynn, Peter
DOI：
10.1073/pnas.80.22.6785
发表时间：
1983-01-01
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES
影响因子：
--
作者：
SADLER, JR;SASMOR, H;BETZ, JL
通讯作者：
BETZ, JL
影响因子：
4.8
作者：
EGGLESTON, AK;ONEILL, TE;KOWALCZYKOWSKI, SC
通讯作者：
KOWALCZYKOWSKI, SC
影响因子：
48
作者：
Noom, Maarten C.;van den Broek, Bram;Wuite, Gijs J. L.
通讯作者：
Wuite, Gijs J. L.