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Base-pairing interactions mediate many intermolecular target recognition events. Even a single base-pair mismatch can cause a substantial difference in activity but how such changes influence the target search kinetics in vivo is unknown. Here, we use high-throughput sequencing and quantitative super-resolution imaging to probe the mutants of bacterial small RNA, SgrS, and their regulation of  ptsG  mRNA target. Mutations that disrupt binding of a chaperone protein, Hfq, and are distal to the mRNA annealing region still decrease the rate of target association,  k  on , and increase the dissociation rate,  k  off , showing that Hfq directly facilitates sRNA–mRNA annealing in vivo. Single base-pair mismatches in the annealing region reduce  k  on  by 24–31% and increase  k  off  by 14–25%, extending the time it takes to find and destroy the target by about a third. The effects of disrupting contiguous base-pairing are much more modest than that expected from thermodynamics, suggesting that Hfq buffers base-pair disruptions.

Effects of individual base-pairs on in vivo target search and destruction kinetics of bacterial small RNA