Mutations of N1 Riboswitch Affect its Dynamics and Recognition by Neomycin Through Conformational Selection
Short, 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 ap...
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| Language: | English |
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Frontiers Media S.A.
2021-02-01
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| Series: | Frontiers in Molecular Biosciences |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fmolb.2021.633130/full |
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| author | Piotr Chyży Piotr Chyży Marta Kulik Marta Kulik Suyong Re Suyong Re Yuji Sugita Yuji Sugita Yuji Sugita Joanna Trylska |
| author_facet | Piotr Chyży Piotr Chyży Marta Kulik Marta Kulik Suyong Re Suyong Re Yuji Sugita Yuji Sugita Yuji Sugita Joanna Trylska |
| author_sort | Piotr Chyży |
| collection | DOAJ |
| description | Short, 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. |
| first_indexed | 2024-12-20T02:42:16Z |
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| id | doaj.art-1f39e3f8ac554cfd90f337f7f8b86bf6 |
| institution | Directory Open Access Journal |
| issn | 2296-889X |
| language | English |
| last_indexed | 2024-12-20T02:42:16Z |
| publishDate | 2021-02-01 |
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| series | Frontiers in Molecular Biosciences |
| spelling | doaj.art-1f39e3f8ac554cfd90f337f7f8b86bf62022-12-21T19:56:17ZengFrontiers Media S.A.Frontiers in Molecular Biosciences2296-889X2021-02-01810.3389/fmolb.2021.633130633130Mutations of N1 Riboswitch Affect its Dynamics and Recognition by Neomycin Through Conformational SelectionPiotr Chyży0Piotr Chyży1Marta Kulik2Marta Kulik3Suyong Re4Suyong Re5Yuji Sugita6Yuji Sugita7Yuji Sugita8Joanna Trylska9Centre of New Technologies, University of Warsaw, Warsaw, PolandFaculty of Physics, University of Warsaw, Warsaw, PolandCentre of New Technologies, University of Warsaw, Warsaw, PolandDepartment of Chemistry, Biological and Chemical Research Centre, University of Warsaw, Warsaw, PolandRIKEN Center for Biosystems Dynamics Research (BDR), Kobe, JapanNational Institutes of Biomedical Innovation, Health and Nutrition, Ibaraki, JapanRIKEN Center for Biosystems Dynamics Research (BDR), Kobe, JapanRIKEN Cluster for Pioneering Research (CPR), Wako, JapanRIKEN Center for Computational Science, Kobe, JapanCentre of New Technologies, University of Warsaw, Warsaw, PolandShort, 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.https://www.frontiersin.org/articles/10.3389/fmolb.2021.633130/fullRNAriboswitchaminoglycosidesneomycinmolecular dynamics simulationsreplica exchange with solute tempering |
| spellingShingle | Piotr Chyży Piotr Chyży Marta Kulik Marta Kulik Suyong Re Suyong Re Yuji Sugita Yuji Sugita Yuji Sugita Joanna Trylska Mutations of N1 Riboswitch Affect its Dynamics and Recognition by Neomycin Through Conformational Selection Frontiers in Molecular Biosciences RNA riboswitch aminoglycosides neomycin molecular dynamics simulations replica exchange with solute tempering |
| title | Mutations of N1 Riboswitch Affect its Dynamics and Recognition by Neomycin Through Conformational Selection |
| title_full | Mutations of N1 Riboswitch Affect its Dynamics and Recognition by Neomycin Through Conformational Selection |
| title_fullStr | Mutations of N1 Riboswitch Affect its Dynamics and Recognition by Neomycin Through Conformational Selection |
| title_full_unstemmed | Mutations of N1 Riboswitch Affect its Dynamics and Recognition by Neomycin Through Conformational Selection |
| title_short | Mutations of N1 Riboswitch Affect its Dynamics and Recognition by Neomycin Through Conformational Selection |
| title_sort | mutations of n1 riboswitch affect its dynamics and recognition by neomycin through conformational selection |
| topic | RNA riboswitch aminoglycosides neomycin molecular dynamics simulations replica exchange with solute tempering |
| url | https://www.frontiersin.org/articles/10.3389/fmolb.2021.633130/full |
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