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Mutations of N1 Riboswitch Affect its Dynamics and Recognition by Neomycin Through Conformational Selection

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cris.lastimport.scopus2024-02-12T20:09:13Z
dc.abstract.enShort, structured fragments of non-coding mRNA may act as molecular switches upon binding specific ligands, regulating the translation of proteins encoded downstream this mRNA sequence. One switch, called riboswitch N1, is regulated by aminoglycosides such as neomycin. Nucleobase mutations in the apical loop, although distant from the binding pocket, significantly affect neomycin affinity and riboswitch regulatory efficiency. To explain this influence, we conducted molecular dynamics simulations using generalized replica exchange with solute tempering (gREST). Translation assay of a reporter protein in a yeast system shows that mutating A17 to G in the riboswitch apical loop reduces 6-fold the translation regulation efficiency of the mutant. Indeed, simulations of the unbound riboswitch show that G17 frequently stacks with base 7, while base 8 is stabilized towards the binding site in a way that it may interfere with the conformational selection mechanism and decrease riboswitch regulatory activity. In the riboswitch complexes, this single-point A to G mutation disrupts a strong hydrogen bond between nucleotides 5 and 17 and, instead, a new hydrogen bond between residue 17 and neomycin is created. This change forces neomycin to occupy a slightly shifted position in the binding pocket, which increases neomycin flexibility. Our simulations of the U14C mutation suggest that the riboswitch complex with neomycin is more stable if cytosine 14 is protonated. A hydrogen bond between the RNA phosphate and protonated cytosine appears as the stabilizing factor. Also, based on the cell-free translation assay and isothermal titration calorimetry experiments, mutations of nucleotides 14 and 15 affect only slightly the riboswitch ability to bind the ligand and its activity. Indeed, the simulation of the unbound U15A mutant suggests conformations preformed for ligand binding, which may explain slightly higher regulatory activity of this mutant. Overall, our results corroborate the in vivo and in vitro experiments on the N1 riboswitch-neomycin system, detail the relationship between nucleobase mutations and RNA dynamics, and reveal the conformations playing the major role in the conformational selection mechanism.
dc.affiliationUniwersytet Warszawski
dc.contributor.authorKulik, Marta
dc.contributor.authorSugita, Yuji
dc.contributor.authorRe, Suyong
dc.contributor.authorChyży, Piotr
dc.contributor.authorTrylska, Joanna
dc.date.accessioned2024-01-25T13:22:56Z
dc.date.available2024-01-25T13:22:56Z
dc.date.copyright2021-02-18
dc.date.issued2021
dc.description.accesstimeAT_PUBLICATION
dc.description.financeŚrodki finansowe przyznane na realizację projektu w zakresie badań naukowych lub prac rozwojowych
dc.description.versionFINAL_PUBLISHED
dc.description.volume8
dc.identifier.doi10.3389/FMOLB.2021.633130
dc.identifier.urihttps://repozytorium.uw.edu.pl//handle/item/113174
dc.identifier.weblinkhttps://www.frontiersin.org/articles/10.3389/fmolb.2021.633130/full
dc.languageeng
dc.pbn.affiliationchemical sciences
dc.relation.ispartofFrontiers in Molecular Biosciences
dc.relation.pagesart.no. 633130
dc.rightsOther
dc.sciencecloudnosend
dc.subject.enaminoglycosides
dc.subject.enmolecular dynamics simulations
dc.subject.enneomycin
dc.subject.enreplica exchange with solute tempering
dc.subject.enriboswitch
dc.subject.enRNA
dc.titleMutations of N1 Riboswitch Affect its Dynamics and Recognition by Neomycin Through Conformational Selection
dc.typeJournalArticle
dspace.entity.typePublication