小柯机器人

瑞典乌普萨拉大学Sebastian Deindl和Johan Elf小组合作揭示了DNA序列定位过程中DNA表面探索和操纵子脱靶效应。这一研究成果在线发表在2020年6月24日出版的《自然》上。

研究人员通过共振能量转移和荧光相关性测量相结合的方法表征了单个lac阻遏物（LacI）分子在一维阶段DNA寻找过程是如何定位于DNA表面的。为了在微秒级跟踪滑动LacI分子的旋转，研究人员使用了实时单分子共聚焦激光跟踪结合荧光相关光谱（SMCT–FCS）的方法。荧光信号的波动是通过旋转耦合滑动精确揭示的，其中LacI每转横穿大约40个碱基对（bp）。这个距离大大超过了DNA 10.5 bp螺旋的节距，表明滑动蛋白经常跳出DNA凹槽，这将导致目标序列的频繁脱靶。

使用单分子荧光共振能量转移（smFRET）研究人员直接观察到这种脱靶效应。对smFRET和SMCT-FCS数据的组合分析显示，LacI每200-700μs跳一个或两个凹槽（10-20 bp）。该研究数据表明滑动过程中需要速度和精度之间的平衡：非特异性蛋白质与DNA间的弱相互作用是操纵子脱靶的基础，但也可以加快滑动速度。研究人员预期SMCT–FCS可在毫秒级的时间尺度内监测旋转扩散，同时以毫秒的分辨率跟踪单个分子；该方法将适用于许多其他生物相互作用的实时研究，其可将有效观察时间范围提高两个数量级，这有利于更好的观察这些相互作用。

据悉，许多结合特定DNA序列的蛋白质通过三维扩散与非特异性DNA上的一维滑动相结合来寻找基因组。

附：英文原文

Title: DNA surface exploration and operator bypassing during target search

Author: Emil Marklund, Brad van Oosten, Guanzhong Mao, Elias Amselem, Kalle Kipper, Anton Sabantsev, Andrew Emmerich, Daniel Globisch, Xuan Zheng, Laura C. Lehmann, Otto G. Berg, Magnus Johansson, Johan Elf, Sebastian Deindl

Issue&Volume: 2020-06-24

Abstract: Many proteins that bind specific DNA sequences search the genome by combining three-dimensional diffusion with one-dimensional sliding on nonspecific DNA1,2,3,4,5. Here we combine resonance energy transfer and fluorescence correlation measurements to characterize how individual lac repressor (LacI) molecules explore the DNA surface during the one-dimensional phase of target search. To track the rotation of sliding LacI molecules on the microsecond timescale, we use real-time single-molecule confocal laser tracking combined with fluorescence correlation spectroscopy (SMCT–FCS). The fluctuations in fluorescence signal are accurately described by rotation-coupled sliding, in which LacI traverses about 40 base pairs (bp) per revolution. This distance substantially exceeds the 10.5-bp helical pitch of DNA; this suggests that the sliding protein frequently hops out of the DNA groove, which would result in the frequent bypassing of target sequences. We directly observe such bypassing using single-molecule fluorescence resonance energy transfer (smFRET). A combined analysis of the smFRET and SMCT–FCS data shows that LacI hops one or two grooves (10–20 bp) every 200–700 μs. Our data suggest a trade-off between speed and accuracy during sliding: the weak nature of nonspecific protein–DNA interactions underlies operator bypassing, but also speeds up sliding. We anticipate that SMCT–FCS, which monitors rotational diffusion on the microsecond timescale while tracking individual molecules with millisecond resolution, will be applicable to the real-time investigation of many other biological interactions and will effectively extend the accessible time regime for observing these interactions by two orders of magnitude.

DOI: 10.1038/s41586-020-2413-7

Source: https://www.nature.com/articles/s41586-020-2413-7

Nature：《自然》，创刊于1869年。隶属于施普林格·自然出版集团，最新IF：69.504
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