PDF(Pubmed)
Abstract：
RNA polymerases (RNAPs) carry out the first step in the central dogma of molecular biology by transcribing DNA into RNA. Despite their importance, much about how RNAPs work remains unclear, in part because the small (3.4 Angstrom) and fast (~40 ms/nt) steps during transcription were difficult to resolve. Here, we used high-resolution nanopore tweezers to observe the motion of single Escherichia coli RNAP molecules as it transcribes DNA ~1,000 times improved temporal resolution, resolving single-nucleotide and fractional-nucleotide steps of individual RNAPs at saturating nucleoside triphosphate concentrations. We analyzed RNAP during processive transcription elongation and sequence-dependent pausing at the yrbL elemental pause sequence. Each time RNAP encounters the yrbL elemental pause sequence, it rapidly interconverts between five translocational states, residing predominantly in a half-translocated state. The kinetics and force-dependence of this half-translocated state indicate it is a functional intermediate between pre- and post-translocated states. Using structural and kinetics data, we show that, in the half-translocated and post-translocated states, sequence-specific protein-DNA interaction occurs between RNAP and a guanine base at the downstream end of the transcription bubble (core recognition element). Kinetic data show that this interaction stabilizes the half-translocated and post-translocated states relative to the pre-translocated state. We develop a kinetic model for RNAP at the yrbL pause and discuss this in the context of key structural features.
摘要：
RNA聚合酶（RNAP）通过将DNA转录为RNA来实现分子生物学中心法则的第一步。尽管它们很重要,关于RNAP如何工作的大部分仍不清楚，部分是因为转录过程中的小(3.4埃)和快(~40ms/nt)步骤难以解决。这里,我们使用高分辨率纳米孔镊子来观察单个大肠杆菌RNAP分子的运动，因为它转录DNA~1000倍提高了时间分辨率，在饱和核苷三磷酸浓度下解析单个RNAP的单核苷酸和分数核苷酸步骤。我们分析了rbL元素暂停序列中持续转录延伸和序列依赖性暂停过程中的RNAP。每次RNAP遇到yrbL元素暂停序列时，它在五种转位状态之间迅速相互转换，主要居住在半移位状态。这种半移位状态的动力学和力依赖性表明，它是移位前状态和移位后状态之间的功能中间体。利用结构和动力学数据，我们证明，在半移位和移位后状态，序列特异性蛋白质-DNA相互作用发生在RNAP和转录泡下游端的鸟嘌呤碱基（核心识别元件）之间。动力学数据显示,这种相互作用相对于移位前状态稳定了半移位和移位后状态。我们在yrbL暂停时开发了RNAP的动力学模型，并在关键结构特征的背景下进行了讨论。
