Dataset Information

Multi-Site Conformational Exchange in the Synthetic Neomycin-Sensing Riboswitch Studied by ¹⁹ F NMR.

ABSTRACT: The synthetic neomycin-sensing riboswitch interacts with its cognate ligand neomycin as well as with the related antibiotics ribostamycin and paromomycin. Binding of these aminoglycosides induces a very similar ground state structure in the RNA, however, only neomycin can efficiently repress translation initiation. The molecular origin of these differences has been traced back to differences in the dynamics of the ligand:riboswitch complexes. Here, we combine five complementary fluorine based NMR methods to accurately quantify seconds to microseconds dynamics in the three riboswitch complexes. Our data reveal complex exchange processes with up to four structurally different states. We interpret our findings in a model that shows an interplay between different chemical groups in the antibiotics and specific bases in the riboswitch. More generally, our data underscore the potential of ¹⁹ F NMR methods to characterize complex exchange processes with multiple excited states.

SUBMITTER: Overbeck JH

PROVIDER: S-EPMC10952710 | biostudies-literature | 2023 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Multi-Site Conformational Exchange in the Synthetic Neomycin-Sensing Riboswitch Studied by <sup>19</sup> F NMR.

Overbeck Jan H JH Vögele Jennifer J Nussbaumer Felix F Duchardt-Ferner Elke E Kreutz Christoph C Wöhnert Jens J Sprangers Remco R

Angewandte Chemie (International ed. in English) 20230428 23

The synthetic neomycin-sensing riboswitch interacts with its cognate ligand neomycin as well as with the related antibiotics ribostamycin and paromomycin. Binding of these aminoglycosides induces a very similar ground state structure in the RNA, however, only neomycin can efficiently repress translation initiation. The molecular origin of these differences has been traced back to differences in the dynamics of the ligand:riboswitch complexes. Here, we combine five complementary fluorine based NM ...[more]

PMID: 36970768

Similar Datasets

Project 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.

Dataset Information

Multi-Site Conformational Exchange in the Synthetic Neomycin-Sensing Riboswitch Studied by ¹⁹ F NMR.

Publications

Multi-Site Conformational Exchange in the Synthetic Neomycin-Sensing Riboswitch Studied by <sup>19</sup> F NMR.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Dataset Information

Multi-Site Conformational Exchange in the Synthetic Neomycin-Sensing Riboswitch Studied by 19 F NMR.

Publications

Multi-Site Conformational Exchange in the Synthetic Neomycin-Sensing Riboswitch Studied by <sup>19</sup> F NMR.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Multi-Site Conformational Exchange in the Synthetic Neomycin-Sensing Riboswitch Studied by ¹⁹ F NMR.