Growing RNAs fold differently as they are transcribed, which modulates their finally adopted structures. Riboswitches regulate gene expression by structural change, which are sensitive to co-transcriptionally structural biology. Here we develop a strategy to track the structural change of RNAs during transcription at single-nucleotide and single-molecule resolution and use it to monitor individual transcripts of the SAM-VI riboswitch (riboSAM) as transcription proceeds, observing co-existence of five states in riboSAM. We report a bifurcated helix in one newly identified state from NMR and single-molecule FRET (smFRET) results, and its presence directs the translation inhibition in our cellular translation experiments. A model is proposed to illustrate the distinct switch patterns and gene-regulatory outcome of riboSAM when SAM is present or absent. Our strategy enables the precise mapping of RNAs' conformational landscape during transcription, and may combine with detection methods other than smFRET for structural studies of RNAs in general.
© 2023. The Author(s).