Cargando…
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...
Autores principales: | , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Frontiers Media S.A.
2021
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7942488/ https://www.ncbi.nlm.nih.gov/pubmed/33708793 http://dx.doi.org/10.3389/fmolb.2021.633130 |
_version_ | 1783662326451273728 |
---|---|
author | Chyży, Piotr Kulik, Marta Re, Suyong Sugita, Yuji Trylska, Joanna |
author_facet | Chyży, Piotr Kulik, Marta Re, Suyong Sugita, Yuji Trylska, Joanna |
author_sort | Chyży, Piotr |
collection | PubMed |
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. |
format | Online Article Text |
id | pubmed-7942488 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-79424882021-03-10 Mutations of N1 Riboswitch Affect its Dynamics and Recognition by Neomycin Through Conformational Selection Chyży, Piotr Kulik, Marta Re, Suyong Sugita, Yuji Trylska, Joanna Front Mol Biosci Molecular Biosciences 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. Frontiers Media S.A. 2021-02-18 /pmc/articles/PMC7942488/ /pubmed/33708793 http://dx.doi.org/10.3389/fmolb.2021.633130 Text en Copyright © 2021 Chyży, Kulik, Re, Sugita and Trylska. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Molecular Biosciences Chyży, Piotr Kulik, Marta Re, Suyong Sugita, Yuji Trylska, Joanna Mutations of N1 Riboswitch Affect its Dynamics and Recognition by Neomycin Through Conformational Selection |
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 | Molecular Biosciences |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7942488/ https://www.ncbi.nlm.nih.gov/pubmed/33708793 http://dx.doi.org/10.3389/fmolb.2021.633130 |
work_keys_str_mv | AT chyzypiotr mutationsofn1riboswitchaffectitsdynamicsandrecognitionbyneomycinthroughconformationalselection AT kulikmarta mutationsofn1riboswitchaffectitsdynamicsandrecognitionbyneomycinthroughconformationalselection AT resuyong mutationsofn1riboswitchaffectitsdynamicsandrecognitionbyneomycinthroughconformationalselection AT sugitayuji mutationsofn1riboswitchaffectitsdynamicsandrecognitionbyneomycinthroughconformationalselection AT trylskajoanna mutationsofn1riboswitchaffectitsdynamicsandrecognitionbyneomycinthroughconformationalselection |