Linking folding dynamics and function of SAM/SAH riboswitches at the single molecule level

Author:

Liao Ting-Wei1ORCID,Huang Lin2ORCID,Wilson Timothy J3,Ganser Laura R1,Lilley David M J3,Ha Taekjip145ORCID

Affiliation:

1. Department of Biophysics, Johns Hopkins University , Baltimore, MD  21218, USA

2. Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Guangdong-Hong Kong Joint Laboratory for RNA Medicine, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University , Guangzhou  510120, China

3. Nucleic Acid Structure Research Group, MSI/WTB Complex, The University of Dundee , Dundee , Dow Street, Dundee DD1 5EH, UK

4. Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine , Baltimore, MD 21205, USA

5. Howard Hughes Medical Institute , Baltimore, MD, USA

Abstract

Abstract Riboswitches are regulatory elements found in bacterial mRNAs that control downstream gene expression through ligand-induced conformational changes. Here, we used single-molecule FRET to map the conformational landscape of the translational SAM/SAH riboswitch and probe how co-transcriptional ligand-induced conformational changes affect its translation regulation function. Riboswitch folding is highly heterogeneous, suggesting a rugged conformational landscape that allows for sampling of the ligand-bound conformation even in the absence of ligand. The addition of ligand shifts the landscape, favoring the ligand-bound conformation. Mutation studies identified a key structural element, the pseudoknot helix, that is crucial for determining ligand-free conformations and their ligand responsiveness. We also investigated ribosomal binding site accessibility under two scenarios: pre-folding and co-transcriptional folding. The regulatory function of the SAM/SAH riboswitch involves kinetically favoring ligand binding, but co-transcriptional folding reduces this preference with a less compact initial conformation that exposes the Shine–Dalgarno sequence and takes min to redistribute to more compact conformations of the pre-folded riboswitch. Such slow equilibration decreases the effective ligand affinity. Overall, our study provides a deeper understanding of the complex folding process and how the riboswitch adapts its folding pattern in response to ligand, modulates ribosome accessibility and the role of co-transcriptional folding in these processes.

Funder

US National Institutes of Health

Cancer Research UK

EPSRC

National Natural Science Foundation of China

Guangdong Science and Technology Department

Howard Hughes Medical Institute

Publisher

Oxford University Press (OUP)

Subject

Genetics

Cited by 1 articles. 订阅此论文施引文献 订阅此论文施引文献,注册后可以免费订阅5篇论文的施引文献,订阅后可以查看论文全部施引文献

1. Fluorescence resonance energy transfer at the single-molecule level;Nature Reviews Methods Primers;2024-03-28

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