The drifting biochemical basis of an essential molecular interaction

Essential biochemical activities are thought to rest on optimized molecular mechanisms that remain conserved during evolution. We traced how the mode of interaction between primary microRNAs and their processing enzyme changed across 460 million years of vertebrate evolution. Through dense phylogenetic reconstruction and high-throughput in vitro processing, we quantified the processing rates of over a thousand ancestral and extant primary microRNAs and all of their possible point mutants. Historical substitutions continuously remodeled the interaction while preserving processing rate. By redistributing base pairs, wobbles, and mismatches and inducing conformational rearrangements, they slightly strengthened or weakened random parts of the interface, gradually reshaping the pattern and biochemical basis of affinity. Each substitution also altered the effects of possible mutations, randomizing the pattern of mutational tolerance within 10%-20% sequence divergence. Overall, the molecular basis of primary microRNA processing is not a fixed outcome of evolutionary optimization but an ever-changing product of drift.