Department of Biochemistry and Molecular Biology, Center for RNA Molecular Biology, 203 Althouse, Pennsylvania State University, University Park, PA 16802, USA.
Department of Chemistry, 260 Engineering and Science, University of Pittsburgh at Johnstown, Johnstown, PA 15904, USA.
Nucleic Acids Res. 2024 Nov 27;52(21):12945-12960. doi: 10.1093/nar/gkae981.
Transcription by RNA polymerase is punctuated by transient pausing events. Pausing provides time for RNA folding and binding of regulatory factors to the paused elongation complex. We previously identified 1600 NusG-dependent pauses throughout the Bacillus subtilis genome, with ∼20% localized to 5' leader regions, suggesting a regulatory role for these pauses. We examined pauses associated with known riboswitches to determine whether pausing is a common feature of these mechanisms. NusG-dependent pauses in the fmnP, tenA, mgtE, lysP and mtnK riboswitches were in strategic positions preceding the critical decision between the formation of alternative antiterminator or terminator structures, which is a critical feature of transcription attenuation mechanisms. In vitro transcription assays demonstrated that pausing increased the frequency of termination in the presence of the cognate ligand. NusG-dependent pausing also reduced the ligand concentration required for efficient termination. In vivo expression studies with transcriptional fusions confirmed that NusG-dependent pausing is a critical component of each riboswitch mechanism. Our results indicate that pausing enables cells to sense a broader range of ligand concentrations for fine-tuning riboswitch attenuation mechanisms.
RNA 聚合酶转录会被短暂的暂停事件打断。暂停为 RNA 折叠和调节因子与暂停延伸复合物结合提供了时间。我们之前在枯草芽孢杆菌基因组中鉴定了 1600 个依赖 NusG 的暂停，其中约 20%定位于 5' 先导区，这表明这些暂停具有调节作用。我们研究了与已知的核糖开关相关的暂停，以确定暂停是否是这些机制的共同特征。在 fmnP、tenA、mgtE、lysP 和 mtnK 核糖开关中，依赖 NusG 的暂停位于形成替代终止子或终止子结构的关键决策之前的战略位置，这是转录衰减机制的一个关键特征。体外转录实验表明，在存在同源配体的情况下，暂停增加了终止的频率。依赖 NusG 的暂停还降低了有效终止所需的配体浓度。与转录融合的体内表达研究证实，依赖 NusG 的暂停是每个核糖开关机制的关键组成部分。我们的结果表明，暂停使细胞能够感知更宽范围的配体浓度，从而精细调整核糖开关衰减机制。
