Stress Localization in the RNA Backbone: A Mechanical Footprint for Predicting Base-Backbone Tertiary Contacts

A physico-chemical basis to understand the site-specificity of intramolecular nucleophilic attack in RNA self-splicing involves the identification of vulnerable backbone regions in addition to determining the proper placement of attacking groups. In preliminary relevant work we have introduced the decisive concept of structure-induced localized absorption of stress by backbone degrees of freedom. In this way we implemented a mechanical approach which incorporates the consensus structural information as a constraint and correctly identifies reactive sites as strain hot spots. In this work we turn this approach into a predictive tool to search for structural constraints which are necessary to localize strain at pre-determined splicing and cyclization sites. In particular, we identify tertiary base backbone contacts regarding them as appropriate constraints to the backbone mechanics. To implement our approach we introduce an effective Hamiltonian which governs the exploration of backbone conformation space by energetically penalizing structural distortions. We show how this Hamiltonian singles out specific regions of stress associated with reactive sites. As an illustration, we apply this working principle to a specific ribozyme, the cobI5 intron, for which the tertiary interactions predicted to be functional in 3′ splicing have not been previously determined experimentally. To establish the predictive value of our approach, we identify the tertiary contacts that should be present in the cobI5 intron to serve as scaffolds stabilizing the conserved P10 secondary interaction and to introduce ribose conformational rigidity necessary to localize strain precisely at the 3′ splicing site. Guided by our computations, the purported interactions are confirmed using deoxyribose substitution probes.