Inlow Koe, Tenenbaum Debora, Friedman Larry J, Kondev Jane, Gelles Jeff
Department of Biochemistry, Brandeis University, Waltham, MA 02453, USA.
Department of Physics, Brandeis University, Waltham, MA 02453, USA.
bioRxiv. 2023 Mar 24:2023.03.22.533849. doi: 10.1101/2023.03.22.533849.
Free-living bacteria have regulatory systems that can quickly reprogram gene transcription in response to changes in cellular environment. The RapA ATPase, a prokaryotic homolog of the eukaryote Swi2/Snf2 chromatin remodeling complex, may facilitate such reprogramming, but the mechanisms by which it does so is unclear. We used multi-wavelength single-molecule fluorescence microscopy in vitro to examine RapA function in the transcription cycle. In our experiments, RapA at < 5 nM concentration did not appear to alter transcription initiation, elongation, or intrinsic termination. Instead, we directly observed a single RapA molecule bind specifically to the kinetically stable post-termination complex (PTC) -- consisting of core RNA polymerase (RNAP) bound to dsDNA -- and efficiently remove RNAP from DNA within seconds in an ATP-hydrolysis-dependent reaction. Kinetic analysis elucidates the process through which RapA locates the PTC and the key mechanistic intermediates that bind and hydrolyze ATP. This study defines how RapA participates in the transcription cycle between termination and initiation and suggests that RapA helps set the balance between global RNAP recycling and local transcription re-initiation in proteobacterial genomes.
RNA synthesis is an essential conduit of genetic information in all organisms. After transcribing an RNA, the bacterial RNA polymerase (RNAP) must be reused to make subsequent RNAs, but the steps that enable RNAP reuse are unclear. We directly observed the dynamics of individual molecules of fluorescently labeled RNAP and the enzyme RapA as they colocalized with DNA during and after RNA synthesis. Our studies show that RapA uses ATP hydrolysis to remove RNAP from DNA after the RNA is released from RNAP and reveal essential features of the mechanism by which this removal occurs. These studies fill in key missing pieces in our current understanding of the events that occur after RNA is released and that enable RNAP reuse.
自由生活的细菌具有调节系统，可根据细胞环境的变化快速重新编程基因转录。RapA ATP酶是真核生物Swi2/Snf2染色质重塑复合体的原核同源物，可能促进这种重新编程，但其具体机制尚不清楚。我们在体外使用多波长单分子荧光显微镜来研究RapA在转录周期中的功能。在我们的实验中，浓度低于5 nM的RapA似乎不会改变转录起始、延伸或内在终止。相反，我们直接观察到单个RapA分子特异性结合动力学稳定的终止后复合体（PTC）——由与双链DNA结合的核心RNA聚合酶（RNAP）组成——并在数秒内通过ATP水解依赖性反应从DNA上有效去除RNAP。动力学分析阐明了RapA定位PTC的过程以及结合和水解ATP的关键机制中间体。这项研究定义了RapA如何参与终止和起始之间的转录周期，并表明RapA有助于在变形菌基因组中建立全局RNAP循环和局部转录重新起始之间的平衡。
RNA合成是所有生物体中遗传信息的重要传递途径。转录RNA后，细菌RNA聚合酶（RNAP）必须被重新利用以合成后续的RNA，但使RNAP重新利用的步骤尚不清楚。我们直接观察了荧光标记的RNAP和酶RapA的单个分子在RNA合成期间和之后与DNA共定位时的动态。我们的研究表明，RapA在RNA从RNAP释放后利用ATP水解从DNA上去除RNAP，并揭示了这种去除发生机制的基本特征。这些研究填补了我们目前对RNA释放后发生的、使RNAP重新利用的事件理解中的关键缺失部分。
