Single-molecule FRET observes opposing effects of urea and TMAO on structurally similar meso- and thermophilic riboswitch RNAs

Author:

Hou Qian12ORCID,Chatterjee Surajit13,Lund Paul E1,Suddala Krishna C14,Walter Nils G1ORCID

Affiliation:

1. Single Molecule Analysis Group, Department of Chemistry, University of Michigan , Ann Arbor , MI  48109 , USA

2. Tri-Institutional PhD Program in Chemical Biology, Weill Cornell Medicine, The Rockefeller University, Memorial Sloan Kettering Cancer Center , NY , NY  10021 , USA

3. Department of Physics, Case Western Reserve University , Cleveland, Ohio 44106, USA

4. Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, NIH , Bethesda , MD  20892 , USA

Abstract

Abstract Bacteria live in a broad range of environmental temperatures that require adaptations of their RNA sequences to maintain function. Riboswitches are regulatory RNAs that change conformation upon typically binding metabolite ligands to control bacterial gene expression. The paradigmatic small class-I preQ1 riboswitches from the mesophile Bacillus subtilis (Bsu) and the thermophile Thermoanaerobacter tengcongensis (Tte) adopt similar pseudoknot structures when bound to preQ1. Here, we use UV-melting analysis combined with single-molecule detected chemical denaturation by urea to compare the thermodynamic and kinetic folding properties of the two riboswitches, and the urea-countering effects of trimethylamine N-oxide (TMAO). Our results show that, first, the Tte riboswitch is more thermotolerant than the Bsu riboswitch, despite only subtle sequence differences. Second, using single-molecule FRET, we find that urea destabilizes the folded pseudoknot structure of both riboswitches, yet has a lower impact on the unfolding kinetics of the thermodynamically less stable Bsu riboswitch. Third, our analysis shows that TMAO counteracts urea denaturation and promotes folding of both the riboswitches, albeit with a smaller effect on the more stable Tte riboswitch. Together, these findings elucidate how subtle sequence adaptations in a thermophilic bacterium can stabilize a common RNA structure when a new ecological niche is conquered.

Funder

National Institutes of Health

Department of Chemistry

Publisher

Oxford University Press (OUP)

Subject

Genetics

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