Stephen Christine, Palmer Danea E, Bautista Clarisa, Mishanina Tatiana V
Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, CA 92093, United States.
Nucleic Acids Res. 2025 Jun 6;53(11). doi: 10.1093/nar/gkaf477.
Manganese (Mn)-sensing riboswitches protect bacteria from Mn toxicity by upregulating expression of Mn exporters. The Mn aptamers share key features but diverge in other important elements, including within the metal-binding core. Although X-ray crystal structures of isolated aptamers exist, these structural snapshots lack crucial details about how the aptamer communicates the presence or absence of ligand to the expression platform. In this work, we investigated the Mn-sensing translational riboswitches in Escherichia coli (mntP and alx), which differ in aptamer secondary structure, nucleotide sequence, and pH-dependence of Mn response. We performed co-transcriptional RNA chemical probing, allowing us to visualize RNA folding intermediates that form and resolve en route to the final folded riboswitch. For the first time, we report that sampling of metal ions by the RNA begins before the aptamer synthesis and folding are complete. At a single-nucleotide resolution, we pinpoint the transcription window where "riboswitching" occurs in response to Mn binding and uncover key differences in how the alx and mntP riboswitches fold. Finally, we describe riboswitch-specific effects of pH, providing insights into how two members of the same riboswitch family differentially sense two distinct environmental cues: concentration of Mn and pH.
锰（Mn）感应核糖开关通过上调锰输出蛋白的表达来保护细菌免受锰毒性的影响。锰适体具有关键特征，但在其他重要元素上存在差异，包括金属结合核心内部。尽管存在分离适体的X射线晶体结构，但这些结构快照缺乏关于适体如何将配体的存在或不存在传递给表达平台的关键细节。在这项工作中，我们研究了大肠杆菌中的锰感应翻译核糖开关（mntP和alx），它们在适体二级结构、核苷酸序列和锰反应的pH依赖性方面存在差异。我们进行了共转录RNA化学探测，使我们能够可视化在最终折叠的核糖开关形成和解析过程中形成的RNA折叠中间体。我们首次报告，RNA对金属离子的采样在适体合成和折叠完成之前就开始了。在单核苷酸分辨率下，我们确定了响应锰结合发生“核糖开关”的转录窗口，并揭示了alx和mntP核糖开关折叠方式的关键差异。最后，我们描述了pH对核糖开关的特异性影响，深入了解了同一核糖开关家族的两个成员如何不同地感知两种不同的环境线索：锰浓度和pH。
