Helix-length compensation studies reveal the adaptability of the VS ribozyme architecture

Résumé

Compensatory mutations in RNA are generally regarded as those that maintain base pairing, and their identification forms the basis of phylogenetic predictions of RNA secondary structure. However, other types of compensatory mutations can provide higher-order structural and evolutionary information. Here, we present a helix-length compensation study for investigating structure–function relationships in RNA. The approach is demonstrated for stem-loop I and stem-loop V of the Neurospora VS ribozyme, which form a kissing–loop interaction important for substrate recognition. To rapidly characterize the substrate specificity (kcat/KM) of several substrate/ribozyme pairs, a procedure was established for simultaneous kinetic characterization of multiple substrates. Several active substrate/ribozyme pairs were identified, indicating the presence of limited substrate promiscuity for stem Ib variants and helix-length compensation between stems Ib and V. 3D models of the I/V interaction were generated that are compatible with the kinetic data. These models further illustrate the adaptability of the VS ribozyme architecture for substrate cleavage and provide global structural information on the I/V kissing–loop interaction. By exploring higher-order compensatory mutations in RNA our approach brings a deeper understanding of the adaptability of RNA structure, while opening new avenues for RNA research.,

Publication
Nucleic Acids Research
Nicolas Girard
Nicolas Girard
Étudiant au doctorat en bio-informatique (2009-2016 avec Pascale Legault, biochimie et médecine moléculaire)
Sébastien Lemieux
Sébastien Lemieux
Chercheur principal

Chercheur principal, Unité de recherche en bio-informatique fonctionnelle et structurale, IRIC | Direction scientifique de la plateforme de Bio-informatique | Professeur agrégé, Département de biochimie et médecine moléculaire, Université de Montréal