Trapped water molecules are essential to structural dynamics and function of a ribozyme.

Ribozymes are catalytically competent examples of highly structured noncoding RNAs, which are ubiquitous in the processing and regulation of genetic information. Combining explicit-solvent molecular dynamics simulation and single molecule fluorescence spectroscopy approaches, we find that a ribozyme from a subviral plant pathogen exhibits a coupled hydrogen bonding network that communicates dynamic structural rearrangements throughout the catalytic core in response to site-specific chemical modification. Trapped long-residency water molecules are critical for this network and only occasionally exchange with bulk solvent as they pass through a breathing interdomain base stack. These highly structured water molecules line up in a string that may potentially also be involved in specific base catalysis. Our observations suggest important, still underappreciated roles for specifically bound water molecules in the structural dynamics and function of noncoding RNAs.