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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.
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http://dx.doi.org/10.1038/s41467-023-38042-2 | DOI Listing |
Biomolecules
June 2025
Institute of Translational Medicine, School of Medicine, Yangzhou University, Yangzhou 225001, China.
Riboswitches regulate gene expression through intricate dynamic conformational transitions, with divalent cation Mg and their ligands playing pivotal roles in this process. The dynamic structural mechanism by which the S-adenosyl-L-methionine (SAM) responsive SAM-VI riboswitch (riboSAM) regulates the downstream SAM synthase gene translation remains unclear. In this study, we employed position-selective labeling of RNA (PLOR) to incorporate Cy3-Cy5 into designated positions of riboSAM, applying single-molecule Förster resonance energy transfer (smFRET) method to track its conformational switches in response to Mg and SAM.
View Article and Find Full Text PDFJ Med Chem
April 2025
Institute of Pharmaceutical and Biomedical Sciences, Johannes Gutenberg-University, Staudingerweg 5, 55128 Mainz, Germany.
Targeting RNA with small molecules is an emerging field in medicinal chemistry. However, highly potent ligands are often challenging to achieve. One intuitive strategy to enhance ligand's potency is the implementation of positively charged moieties to interact with the negatively charged RNA phosphate backbone.
View Article and Find Full Text PDFInt J Mol Sci
March 2025
Institute of Translational Medicine, School of Medicine, Yangzhou University, Yangzhou 225001, China.
Precisely fluorescently labeling specific nucleotide sites of RNA is critical for gaining insights into the structure and function of RNA through multiple fluorescence detection techniques. The position-selective labeling of RNA (PLOR) method provides a promising strategy to achieve this, wherein the fluorophore-modified NTPs can be co-transcriptionally introduced to specific sites of nascent RNA by using T7 RNA polymerase (T7 RNAP). However, due to steric hindrance limitations, the efficiency of T7 RNAP in recognizing and incorporating large fluorophore-modified NTPs into RNA is far from satisfactory.
View Article and Find Full Text PDFBiomolecules
June 2024
College of Biological and Medical Engineering, Donghua University, Shanghai 201620, China.
The SAM-VI riboswitch undergoes dynamic conformational changes that modulate downstream gene expression. Traditional structural methods such as crystallography capture the bound conformation at high resolution, and additional efforts would reveal details from the dynamic transition. Here, we revealed a transcription-dependent conformation model for SAM-VI riboswitch.
View Article and Find Full Text PDFNat Commun
April 2023
State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, China.
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.
View Article and Find Full Text PDF