Exploring the mechanisms behind the E. coli pH-responsive riboswitch.

Abstract

Riboswitches are 5’-untranslated regions of mRNA that change their conformation in response to ligand binding, dictating whether the downstream gene is transcribed and/or translated into protein. A “pH-responsive element” (PRE) riboswitch discovered in E. coli, however, does not require a ligand binding event in a traditional sense to rearrange its structure and instead senses the environmental H+ concentration. Specifically, at neutral pH, the PRE folds into a translationally inactive structure with an occluded ribosome binding sequence (RBS), whereas at alkaline pH, the PRE adopts a translationally active structure with an exposed RBS. The precise mechanism that controls pH-responsive riboswitching has remained unclear, with two proposed models 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 alkaline pH enhances RNAP pause longevity during PRE synthesis to drive folding of structural motifs in the active conformer. I investigated this proposal by performing the first rigorous kinetic study of E. coli RNAP pausing at key sites for PRE folding under different pH conditions. I find that pH influences RNAP pausing, but not in the proposed manner; instead, my experiments uncovered that alkaline pH either decreases or has no effect on RNAP pause longevity. These results indicate that modulation of RNAP pausing is not the sole mechanism by which pH affects PRE folding and invite the possibility that the riboswitch RNA is actively involved in sensing the cytosolic pH (Stephen and Mishanina, J. Biol. Chem. 2022, 298:102302). I am currently conducting chemical probing and cryo-EM studies of PRE structure to explore the pH-induced RNA structural changes in rigorous detail, which will ultimately illuminate the mechanism for how pH-dependent riboswitching occurs.