Data associated with the publication: Co-transcriptional folding of the glmS ribozyme enables a rapid response to metabolite

This dataset is related to a manuscript submitted to Nucleic Acids Research, describing co-transcriptional folding of the Bacillus subtilis glmS ribozyme, using single molecule total internal reflection fluorescence microscopy. The glmS ribozyme riboswitch, located in the 5’ UTR of the Bacillus subtilis glmS mRNA, regulates cell wall biosynthesis through ligand-induced self-cleavage and decay of the glmS mRNA. Although self-cleavage of the refolded glmS ribozyme has been studied extensively, the kinetics of ribozyme folding and ligand binding during transcription is not understood. Here, we combine TIRF single-molecule assays with kinetic modeling to show that self-cleavage can occur during transcription before the ribozyme is fully synthesized. Moreover, co-transcriptional folding of the RNA at a physiological elongation rate allows the ribozyme core itself to become reactive. DMS footprinting further revealed how slow sequential folding favors formation of the native core structure through fraying of misfolded helices and nucleation of a native pseudoknot. Ribozyme self-cleavage at an early stage of transcription may benefit glmS regulation in B. subtilis, as it exposes the mRNA to exoribonuclease before translation starts. Our results emphasize the importance of co-transcriptional folding of RNA tertiary structure for cis-regulation of mRNA stability.