Comprehensive Sequence-to-Function Mapping of Ligand-Dependent RNA Catalysis

Abstract

Massively-parallel, quantitative measurements of biochemical activity have the potential to revolutionize our understanding of biomolecular sequence-structure-function relationships. We report the development of a novel RNA array assay which enables such measurements to be performed on self-cleaving ribozymes, allowing determination of real-time cleavage kinetics of tens of thousands of sequence variants in a single experiment. We applied this methodology to the glmS ribozyme riboswitch, which performs self-cleavage in a ligand-dependent manner in order to regulate bacterial cell wall synthesis. We determined the cleavage rates of all single and double mutants of this RNA under a range of regulatory ligand concentrations, enabling the calculation of kcat and KM for active variants. This trove of quantitative kinetic parameters across the mutational landscape enabled a number of further analyses. Comparing single-mutant activities with naturally-occurring sequence variation at each position, we found that conservation is driven by cleavage rate, and not by ligand binding affinity. Comprehensive double-mutant measurements permitted a global analysis of rescue interactions, which revealed the functional relevance of several known tertiary contacts, as well as providing evidence for a number of compensatory secondary and tertiary structural rearrangements. Double-mutant kinetics and rescue interactions also provided a combined structural and functional mapping of the ribozyme which successfully identified the full secondary structure at nucleotide resolution, while additionally revealing relative structural and catalytic contributions of individual residues. We believe that our approach will be broadly applicable and extensible, enabling a deeper understanding of sequence-structure-function relationships across a variety of functional RNAs.