Cellular and Molecular Biotechnology Research Institute, National Institute of Advanced Industrial Science and Technology, Tokyo, Japan.
Research Institute for Measurement and Analytical Instrumentation, National Institute of Advanced Industrial Science and Technology, Tsukuba, Japan.
Genes Cells. 2021 Feb;26(2):56-64. doi: 10.1111/gtc.12822. Epub 2020 Dec 19.
During transcription in cells, the transcription complex consisting of RNA polymerase, DNA and nascent RNA is exposed to fluctuating temperature and pressure. However, little is known about the mechanism of transcriptional homeostasis under fluctuating physical parameters. In this study, we generated these fluctuating parameters using pulsed local heating and acoustic waves in the reaction system of transcription by Escherichia coli RNA polymerase, using a terahertz free-electron laser. We demonstrated that transcription processes, including abortive initiation and elongation pausing, and the fidelity of elongation are significantly affected by the laser-based local perturbations. We also found that all these functional alternations in the transcription process are almost completely mitigated by the presence of Gre proteins. It is well known that Gre proteins enhance RNA cleavage of polymerase by binding to the pore structure termed secondary channel. Recently, the chaperone activities have also been proposed for Gre proteins, yet the details directly associated with transcription are largely unknown. Our finding indicates that Gre proteins are necessary for maintaining transcriptional homeostasis under thermal and mechanical stresses.
在细胞内的转录过程中,由 RNA 聚合酶、DNA 和新生 RNA 组成的转录复合物会暴露于不断变化的温度和压力下。然而,人们对于在不断变化的物理参数下转录的动态平衡的机制知之甚少。在这项研究中,我们使用太赫兹自由电子激光在大肠杆菌 RNA 聚合酶的转录反应系统中产生了这些波动的参数,使用局部热脉冲和声波。我们证明了转录过程,包括起始的夭折和延伸暂停,以及延伸的保真度,都受到基于激光的局部扰动的显著影响。我们还发现,转录过程中的所有这些功能变化几乎都可以通过 Gre 蛋白的存在得到缓解。众所周知,Gre 蛋白通过与称为次级通道的孔结构结合来增强聚合酶对 RNA 的切割。最近,也提出了 Gre 蛋白的伴侣活性,但与转录直接相关的细节在很大程度上仍是未知的。我们的发现表明,Gre 蛋白对于维持热和机械应激下的转录动态平衡是必需的。