Jensen Drake, Manzano Ana Ruiz, Rector Maxwell, Tomko Eric J, Record M Thomas, Galburt Eric A
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Saint Louis, MO, 63108, USA.
Department of Biochemistry, University of Wisconsin, Madison, WI, 53706, USA.
bioRxiv. 2023 Mar 13:2023.03.13.532464. doi: 10.1101/2023.03.13.532464.
The first step in gene expression is the transcription of DNA sequences into RNA. Regulation at the level of transcription leads to changes in steady-state concentrations of RNA transcripts, affecting the flux of downstream functions and ultimately cellular phenotypes. Changes in transcript levels are routinely followed in cellular contexts via genome-wide sequencing techniques. However, mechanistic studies of transcription have lagged with respect to throughput. Here, we describe the use of a real-time, fluorescent-aptamer-based method to quantitate steady-state transcription rates of the RNA polymerase. We present clear controls to show that the assay specifically reports on promoter-dependent, full-length RNA transcription rates that are in good agreement with the kinetics determined by gel-resolved, α- P NTP incorporation experiments. We illustrate how the time-dependent changes in fluorescence can be used to measure regulatory effects of nucleotide concentrations and identity, RNAP and DNA concentrations, transcription factors, and antibiotics. Our data showcase the ability to easily perform hundreds of parallel steady-state measurements across varying conditions with high precision and reproducibility to facilitate the study of the molecular mechanisms of bacterial transcription.
RNA polymerase transcription mechanisms have largely been determined from kinetic and structural biology methods. In contrast to the limited throughput of these approaches, RNA sequencing provides genome-wide measurements but lacks the ability to dissect direct biochemical from indirect genetic mechanisms. Here, we present a method that bridges this gap, permitting high-throughput fluorescence-based measurements of steady-state transcription kinetics. We illustrate how an RNA-aptamer-based detection system can be used to generate quantitative information on direct mechanisms of transcriptional regulation and discuss the far-reaching implications for future applications.
基因表达的第一步是将DNA序列转录为RNA。转录水平的调控会导致RNA转录本稳态浓度的变化，影响下游功能的通量并最终影响细胞表型。通过全基因组测序技术可常规跟踪细胞环境中转录本水平的变化。然而，转录的机制研究在通量方面滞后。在这里，我们描述了一种基于实时荧光适体的方法来定量RNA聚合酶的稳态转录速率。我们提供了明确的对照，以表明该测定法专门报告依赖启动子的全长RNA转录速率，这与通过凝胶分辨的α-P NTP掺入实验确定的动力学结果高度一致。我们说明了如何利用荧光随时间的变化来测量核苷酸浓度和同一性、RNA聚合酶和DNA浓度、转录因子及抗生素的调控作用。我们的数据展示了能够在不同条件下轻松进行数百次平行稳态测量的能力，具有高精度和可重复性，有助于研究细菌转录的分子机制。
RNA聚合酶转录机制在很大程度上是通过动力学和结构生物学方法确定的。与这些方法通量有限形成对比的是，RNA测序可提供全基因组测量，但缺乏区分直接生化机制和间接遗传机制的能力。在这里，我们提出了一种弥合这一差距的方法，允许基于荧光的高通量稳态转录动力学测量。我们说明了基于RNA适体的检测系统如何用于生成有关转录调控直接机制的定量信息，并讨论了对未来应用的深远影响。
Zotero 插件
