Fast target recognition by bacterial sRNA and their Hfq chaperone

Sarah Woodson, Ewelina Malecka, Subrata Panja, Andrew Santiago-Frangos

Johns Hopkins University,

Bacterial small non-coding RNAs (sRNAs) regulate the expression of many target mRNAs against a background of myriad competing RNA and protein interactions. The recognition of mRNA targets is aided by the chaperone protein Hfq that facilitates sRNA-mRNA base pairing. A member of the Sm/Lsm family, Hfq uses distinct RNA binding surfaces to recognize a diverse family of sRNAs. To understand how Hfq’s multiple binding surfaces accelerate sRNA-mRNA annealing, we developed a TIRF-based single-molecule fluorescence platform to visualize Hfq-mediated sRNA-mRNA annealing process in real time. Using this platform, distinct events which take place before and during the formation of the Hfq-mRNA-sRNA ternary complex can be observed. Moreover, our assay allows for a direct observation of the helix formation initiation and shows that sRNA-mRNA annealing can be achieved via multiple pathways. We find that the likelihood of successful target recognition depends on the RNA secondary structure and its interaction with more than one Hfq surface, explaining why certain classes of sRNAs regulate their targets more efficiently than others.

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