Cotranscriptional folding of a 5́′ stem-loop in the Escherichia coli tbpA riboswitch at single-nucleotide resolution

Transcription elongation is one of the most important processes in the cell. During RNA polymerase elongation, the folding of nascent transcripts plays crucial roles in the genetic decision. Bacterial riboswitches are prime examples of RNA regulators that control gene expression by altering their structure upon metabolite sensing. It was previously revealed that the thiamin pyrophosphate-sensing tbpA riboswitch in Escherichia coli cotranscriptionally adopts three main structures leading to metabolite sensing. Here, using single-molecule FRET, we characterize the transition in which the first nascent structure, a 5′ stem-loop, is unfolded during transcription elongation to form the ligand-binding competent structure. Our results suggest that the structural transition occurs in a relatively abrupt manner, i.e., within a 1-2 nucleotide window. Furthermore, a highly dynamic structural exchange is observed, indicating that riboswitch transcripts perform rapid sampling of nascent co-occurring structures. We also observe that the presence of the RNAP stabilizes the 5′ stem-loop along the elongation process, consistent with RNAP interacting with the 5′ stem-loop. Our study emphasizes the role of early folding stem-loop structures in the cotranscriptional formation of complex RNA molecules involved in genetic regulation.