Two riboswitch classes that share a common ligand-binding fold show major differences in the ability to accommodate...

Area of Science:

• Molecular Biology
• RNA Biology
• Structural Biology

Background:

• Riboswitches are RNA molecules that regulate gene expression in response to small molecule binding.
• Prequeuosine1 (preQ1)-sensing riboswitches exist in three distinct classes (I, II, and III) with different structures.
• Class II and III riboswitches utilize conserved nucleotides for preQ1 metabolite binding, but class II is sensitive to binding-pocket mutations.

Purpose of the Study:

• To investigate the impact of mutations on the structure, function, and preQ1 binding affinity of class III riboswitches.
• To compare the mutational tolerance of class III riboswitches with previously studied class II variants.
• To elucidate the molecular mechanisms underlying compensatory interactions and their role in maintaining riboswitch function.

Main Methods:

• Introduction of four equivalent mutations into a class III riboswitch.
• Co-crystal structure determination of mutant class III riboswitches.
• Chemical modification analysis and molecular dynamics (MD) simulations.
• Assessment of gene-regulatory activity in bacteria.
• Principal component analysis (PCA) of MD trajectories.

Main Results:

• Mutations in the class III riboswitch maintained tight preQ1 binding, contrasting with class II.
• Co-crystal structures revealed compensatory interactions that preserved the riboswitch fold.
• MD simulations indicated mutations were not overtly destabilizing, despite localized flexibility changes.
• Class III mutants retained gene-regulatory activity, with pocket floor mutations better tolerated than wall mutations.
• Deleterious mutations altered long-range correlated motions between the binding pocket and regulatory regions.

Conclusions:

• Compensatory interaction formation is context-dependent within the riboswitch fold.
• Class III riboswitches exhibit greater tolerance to binding-pocket mutations than class II.
• Functionally detrimental mutations can disrupt long-range RNA dynamics crucial for gene regulation.