Stepwise sRNA targeting of structured bacterial mRNAs leads to abortive annealing.

Bacterial small RNAs (sRNAs) regulate the expression of hundreds of transcripts via base pairing mediated by the Hfq chaperone protein. sRNAs and the mRNA sites they target are heterogeneous in sequence, length, and secondary structure. To understand how Hfq can flexibly match diverse sRNA and mRNA pairs, we developed a single-molecule Förster resonance energy transfer (smFRET) platform that visualizes the target search on timescales relevant in cells. Here we show that unfolding of target secondary structure on Hfq creates a kinetic energy barrier that determines whether target recognition succeeds or aborts before a stable anti-sense complex is achieved. Premature dissociation of the sRNA can be alleviated by strong RNA-Hfq interactions, explaining why sRNAs have different target recognition profiles. We propose that the diverse sequences and structures of Hfq substrates create an additional layer of information that tunes the efficiency and selectivity of non-coding RNA regulation in bacteria. [Display omitted] • Single-molecule microscopy tracks how Hfq pairs small RNAs with mRNA targets • Hfq chaperone unfolds mRNA structure during base pairing with a small RNA • Unfolding creates a kinetic energy barrier that can cause base pairing to abort • Strong RNA-Hfq interactions increase complex stability and curtail failed attempts Małecka and Woodson visualize how bacterial small RNAs and Hfq bind mRNAs with single-molecule resolution. Structured targets are unfolded by Hfq during sRNA base pairing but increase the probability that the process will abort. These failures help explain why certain targets are regulated more efficiently than others during a stress response. [ABSTRACT FROM AUTHOR]