Observation of structural switch in nascent SAM-VI riboswitch during transcription at single-nucleotide and single-molecule resolution

Abstract

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.

Ligand Response of Guanidine-IV riboswitch at Single-molecule Level

“…In the transcriptional mimicking assay, the nascent RNA displayed a weaker response to guanidine compared to the post-transcriptional counterparts. This suggests that in the guanidine-IV riboswitch system, the DNA templates and RNA polymerase play a role in RNA folding and guanidine binding, in line with the previous findings for the pre-Q1 and SAM-VI riboswitches (Widom et al, 2018; Xue et al, 2023). Moreover, the guanidine sensitivity of EC-87 indicates that guanidine regulation for the guanidine-IV riboswitch occurs before transcription of the aptamer domain is completed.…”

“…However, Xue et al have reported similarities between T7 and E. coli RNAP in the folding of nascent RNA. Additionally, Lou and Woodson have provided valuable insights into the co-transcriptional folding of the glmS ribozyme using T7 RNAP (Xue et al, 2023; Lou & Woodson, 2024).…”

“…This KL facilitates the dissociation of the terminator and promotes transcriptional read-through (Lenkeit et al, 2020;Salvail et al, 2020). KL or pseudoknot formation upon ligand binding is a common occurrence in various riboswitches, including adenine, guanine, ZTP, pre-Q1, fluoride, and cdi-AMP riboswitches ( (Xue et al, 2023). Despite these previous investigations, there is lack of information regarding the conformational changes and ligand-sensing behavior of the guanidine-IV riboswitch during transcription.…”

“…Landgraf et al utilized NMR and computational modeling to identify the ligand-sensing transcriptional window for the c-di-GMP and c-GAMP riboswitches (Landgraf et al, 2022). Hua et al investigated the co-transcriptional folding of the twister ribozyme and the ZTP riboswitch by mimicking the folding process using helicases to dissociate an RNA-DNA hybrid (Hua et al, 2020;Hua et al, 2018) (Xue et al, 2023). Despite these previous investigations, there is lack of information regarding the conformational changes and ligand-sensing behavior of the guanidine-IV riboswitch during transcription.…”

“…Xue et al . monitored the folding of a growing SAM-VI riboswitch at the single-molecule resolution by FRET (Xue et al, 2023). Despite these previous investigations, there is lack of information regarding the conformational changes and ligand-sensing behavior of the guanidine-IV riboswitch during transcription.…”