Full molecular trajectories of RNA polymerase at single base-pair resolution.
Full molecular trajectories of RNA polymerase at single base-pair resolution.
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DOI：
10.1073/pnas.1719906115
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发表时间：
2018-02-06
影响因子：
11.1
通讯作者：
Bustamante C
中科院分区：
文献类型：
--
作者：
Righini M;Lee A;Cañari-Chumpitaz C;Lionberger T;Gabizon R;Coello Y;Tinoco I Jr;Bustamante C
镊子使科学家以高分辨率遵循分子的动态。我们消除了大多数高分辨率双陷阱光学镊子的主要噪声来源，并开发了单分子测定法和一个自学习算法，以使用此方法来揭示此类电动机的完整轨迹。
近年来，高度稳定的光学镊子系统使分子电动机的动力学表征很高，但是，由于使用较差的噪音，与较低的噪声相关的是，与“降低”相关的差异，许多电动机的运动不能始终如一地解决。 - 陷阱光学使用此仪器，我们实施了一项协议，该协议综合了单个基本对轨迹，该轨迹用于测试Markov Markov模型算法的大型状态空间，以恢复其各个步骤。在连续的聚合酶步骤之间。
Optical tweezers enable scientists to follow the dynamics of molecular motors at high resolution. The ability to discern a motor’s discrete steps reveals important insights on its operation. Some motors operate at the scale of angstroms, rendering the observation of their steps extremely challenging. In some cases, such small steps have been observed sporadically; however, the full molecular trajectories of steps and intervals between steps remain elusive due to instrumental noise. Here, we eliminate the main source of noise of most high-resolution dual-trap optical tweezers and developed both a single-molecule assay and a self-learning algorithm to uncover the full trajectories of such a motor: RNA polymerase. Using this method, a whole new set of experiments becomes possible. In recent years, highly stable optical tweezers systems have enabled the characterization of the dynamics of molecular motors at very high resolution. However, the motion of many motors with angstrom-scale dynamics cannot be consistently resolved due to poor signal-to-noise ratio. Using an acousto-optic deflector to generate a “time-shared” dual-optical trap, we decreased low-frequency noise by more than one order of magnitude compared with conventional dual-trap optical tweezers. Using this instrument, we implemented a protocol that synthesizes single base-pair trajectories, which are used to test a Large State Space Hidden Markov Model algorithm to recover their individual steps. We then used this algorithm on real transcription data obtained in the same instrument to fully uncover the molecular trajectories of Escherichia coli RNA polymerase. We applied this procedure to reveal the effect of pyrophosphate on the distribution of dwell times between consecutive polymerase steps.
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影响因子：
3.4
作者：
Mullner, Fiona E.;Syed, Sheyum;Sigworth, Fred J.
通讯作者：
Sigworth, Fred J.
影响因子：
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作者：
Kerssemakers, Jacob W. J.;Munteanu, E. Laura;Dogterom, Marileen
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Dogterom, Marileen
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作者：
FINER, JT;SIMMONS, RM;SPUDICH, JA
通讯作者：
SPUDICH, JA
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Asbury, CL;Fehr, AN;Block, SM
通讯作者：
Block, SM
影响因子：
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Komissarova, N;Kashlev, M
通讯作者：
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