Abstract
Hundreds of bacterial small RNAs (sRNAs) require the Hfq chaperone to regulate mRNA expression. Hfq is limiting, thus competition among sRNAs for binding to Hfq shapes the proteomes of individual cells. To understand how sRNAs compete for a common partner, we present a single-molecule fluorescence platform to simultaneously visualize binding and release of multiple sRNAs with Hfq. We show that RNA residents rarely dissociate on their own. Instead, clashes between residents and challengers on the same face of Hfq cause rapid exchange, whereas RNAs that recognize different surfaces may cohabit Hfq for several minutes before one RNA departs. The prevalence of these pathways depends on the structure of each RNA and how it interacts with Hfq. We propose that sRNA diversity creates many pairwise interactions with Hfq that allow for distinct biological outcomes: active exchange favors fast regulation whereas co-residence of dissimilar RNAs favors target co-recognition or target exclusion.