V. harveyi uses multiple related Qrr sRNAs, each ∼100 nucleotides in length, to control the expression of several target mRNAs involved in quorum sensing, including those encoded by luxM, luxO, luxR and aphA. To study the mechanisms by which one of the Qrr sRNAs, Qrr3, regulates the expression of these different mRNA targets, Feng et al. carried out competition assays in Escherichia coli involving an inducible Qrr3 sRNA and different combinations of two inducible target mRNAs, in which different coloured fluorescent reporter proteins were encoded downstream of the Qrr3-binding sites in the target mRNAs. The fates of the Qrr3 sRNA and target mRNAs were tracked by Northern blotting, and expression of the encoded fluorescent proteins was monitored by flow cytometry. The investigators identified four distinct modes of action of Qrr3 on different target mRNAs: nucleolytic degradation of the target mRNA (with or without simultaneous degradation of Qrr3), sequestration of the target mRNA, and activation of translation from the target mRNA with concomitant Qrr3 degradation.
Previous experimental work and RNA–RNA binding predictions showed that all of these regulatory modes involve the hybridization of Qrr3 to its targets, so Feng et al. mutated the Qrr-binding sites in the target mRNAs to test what particular features of base-pairing determine the regulatory mode. For example, they showed that when hybridization to the target mRNA disrupts a 5′ hairpin structure in Qrr3, this leaves Qrr3 susceptible to degradation, and that the overall binding strength of the Qrr3–mRNA interaction determines the degree of Qrr3 sequestration.
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