Insights into the cotranscriptional and translational control mechanisms of the Escherichia coli tbpA thiamin pyrophosphate riboswitch

Abstract

Riboswitches regulate gene expression by modulating their structure upon metabolite binding. These RNA orchestrate several layers of regulation to achieve genetic control. Although Escherichia coli riboswitches modulate translation initiation, several cases have been reported where riboswitches also modulate mRNA levels. Here, we characterize the regulation mechanisms of the thiamin pyrophosphate (TPP) tbpA riboswitch in E. coli. Our results indicate that the tbpA riboswitch modulates both levels of translation and transcription and that TPP sensing is achieved more efficiently cotranscriptionally than post-transcriptionally. The preference for cotranscriptional binding is also observed when monitoring the TPP-dependent inhibition of translation initiation. Using single-molecule approaches, we observe that the aptamer domain freely fluctuates between two main structures involved in TPP recognition. Our results suggest that translation initiation is controlled through the ligand-dependent stabilization of the riboswitch structure. This study demonstrates that riboswitch cotranscriptional sensing is the primary determinant in controlling translation and mRNA levels.