Riboswitches are 5’-untranslated regions of mRNA that change their conformation in response to ligand binding, allowing post-transcriptional gene regulation. This ligand-based model of riboswitch function has been expanded with the discovery of a “pH-responsive element” (PRE) riboswitch in E. coli. At neutral pH, the PRE folds into a translationally inactive structure with an occluded ribosome binding sequence, whereas at alkaline pH the PRE adopts a translationally active structure. This unique riboswitch does not rely on ligand binding in a traditional sense to modulate its alternative folding outcomes. Rather, pH controls riboswitch folding by two possible modes that are yet to be distinguished; pH either regulates the transcription rate of RNA polymerase (RNAP) or acts on the RNA itself. Previous work suggested that RNAP pausing is prolonged by alkaline pH at two sites, stimulating PRE folding into the active structure. To date, there has been no rigorous exploration into how pH influences RNAP pausing kinetics during PRE synthesis. To provide that understanding and distinguish between pH acting on RNAP versus RNA, we investigated RNAP pausing kinetics at key sites for PRE folding under different pH conditions. We find that pH influences RNAP pausing, but not in the manner proposed previously. Rather, alkaline pH either decreases or has no effect on RNAP pause longevity, suggesting the modulation of RNAP pausing is not the sole mechanism by which pH affects PRE folding. These findings invite the possibility that the RNA itself actively participates in the sensing of pH.

核糖开关是 mRNA 的 5' 非翻译区，可响应配体结合而改变其构象，从而实现转录后基因调控。这种基于配体的核糖开关功能模型已随着大肠杆菌中“pH 响应元件”（PRE）核糖开关的发现而得到扩展. 在中性 pH 下，PRE 折叠成具有封闭核糖体结合序列的翻译无活性结构，而在碱性 pH 下，PRE 采用翻译活性结构。这种独特的核糖开关不依赖于传统意义上的配体结合来调节其替代折叠结果。相反，pH 通过两种尚未区分的可能模式控制核糖开关折叠；pH 要么调节 RNA 聚合酶 (RNAP) 的转录速率，要么作用于 RNA 本身。以前的工作表明，两个位点的碱性 pH 值会延长 RNAP 的暂停时间，从而刺激 PRE 折叠成活性结构。迄今为止，还没有对 PRE 合成过程中 pH 值如何影响 RNAP 暂停动力学进行严格的探索。为了提供对作用于 RNAP 和 RNA 的 pH 值的理解和区分，我们研究了不同 pH 条件下 PRE 折叠关键位点的 RNAP 暂停动力学。我们发现 pH 值会影响 RNAP 暂停，但不是以之前提出的方式。相反，碱性 pH 降低或对 RNAP 暂停寿命没有影响，这表明 RNAP 暂停的调节不是 pH 影响 PRE 折叠的唯一机制。这些发现提出了 RNA 本身积极参与 pH 传感的可能性。