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bioRxiv · 10.1101/2021.03.12.434875

Ensemble Switching Unveils a Kinetic Rheostat Mechanism of the Eukaryotic Thiamine PyrophosphateRiboswitch

Ma, J.·Saikia, N.·Godar, S.·Hamilton, G. L.·Ding, F.·Alper, J.·Sanabria, H.
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Abstract

Thiamine pyrophosphate (TPP) riboswitches regulate thiamine metabolism by inhibiting the translation of enzymes essential to thiamine synthesis pathways upon binding to thiamine pyrophosphate in cells across all domains of life. Recent work on the Arabidopsis thaliana TPP riboswitch suggests a multi-step TPP binding process involving multiple riboswitch conformational ensembles and that Mg2+ dependence underlies the mechanism of TPP recognition and subsequent transition to the translation-inhibiting state of the switching sequence followed by changes in the expression platform. However, details of the relationship between TPP riboswitch conformational changes and interactions with TPP and Mg2+ in the aptamer domain constituting this mechanism are unknown. Therefore, we integrated single-molecule multiparameter fluorescence and force spectroscopy with atomistic molecular dynamics simulations and found that conformational transitions within the aptamer domain associated with TPP and Mg2+ ligand binding occurred between at least five different ensembles on timescales ranging from s to ms. These dynamics are at least an order of magnitude faster than folding and unfolding kinetics associated with translation-state switching in the switching sequence. Moreover, we propose that two pathways exist for ligand recognition. Together, our results suggest a dynamic ensemble switching of the aptamer domain that may lead to the translation-inhibiting state of the riboswitch. Additionally, our results suggest that multiple configurations could enable inhibitory tuning manifested through ligand-dependent changes via ensemble switching and kinetic rheostat-like behavior of the Arabidopsis thaliana TPP riboswitch.

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Ma, J., Saikia, N., Godar, S., Hamilton, G. L., Ding, F., Alper, J., Sanabria, H.. 2021-03-12. Ensemble Switching Unveils a Kinetic Rheostat Mechanism of the Eukaryotic Thiamine PyrophosphateRiboswitch. https://doi.org/10.1101/2021.03.12.434875

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