The binding of Class II sRNA MgrR to two different sites on matchmaker protein Hfq enables efficient competition for Hfq and annealing to regulated mRNAs.

Joanna Kwiatkowska,Kenneth A Johnson,Mikolaj Olejniczak,Zuzanna Wroblewska

doi:10.1261/rna.067777.118

Joanna Kwiatkowska, Kenneth A Johnson + Show 2 more

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https://doi.org/10.1261/rna.067777.118

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Abstract

MgrR is an Hfq-dependent sRNA, whose transcription is controlled by the level of Mg2+ ions in Escherichia coli. MgrR belongs to Class II sRNAs because its stability in the cell is affected by mutations in Hfq differently than canonical, Class I sRNAs. Here, we examined the effect of mutations in RNA binding sites of Hfq on the kinetics of the annealing of MgrR to two different target mRNAs, eptB and ygdQ, by global data fitting of the reaction kinetics monitored by gel electrophoresis of intermediates and products. The data showed that the mutation on the rim of the Hfq ring trapped MgrR on Hfq preventing the annealing of MgrR to either mRNA. The mutation in the distal face slowed the ternary complex formation and affected the release of MgrR-mRNA complexes from Hfq, while the mutation in the proximal face weakened the MgrR binding to Hfq and in this way affected the annealing. Moreover, competition assays established that MgrR bound to both faces of Hfq and competed against other sRNAs. Further studies showed that uridine-rich sequences located in less structurally stable regions served as Hfq binding sites in each mRNA. Overall, the data show that the binding of MgrR sRNA to both faces of the Hfq ring enables it to efficiently anneal to target mRNAs. It also confers on MgrR a competitive advantage over other sRNAs, which could contribute to efficient cellular response to changes in magnesium homeostasis.

Highlights

Small regulatory RNAs contribute to bacterial response to environmental stress, maintenance of cell homeostasis (Updegrove et al 2015; Wagner and Romby 2015), and the modulation of bacterial virulence (Ellis et al 2017; Heidrich et al 2017)
To understand the impact of the RNA structure on the Hfq contribution to MgrR annealing to its targets, MgrR Small RNAs (sRNAs) was probed with structure-specific RNases (Fig. 1A,B; Supplemental Fig. S1A)
As it has been previously observed (Schu et al 2015), the 98-nt-long MgrR sRNA contains in its central part a long adenosine-rich sequence, which could be considered as an (ARN)5 motif with two mismatches (Fig. 1A,B)

Summary

Introduction

Small regulatory RNAs contribute to bacterial response to environmental stress, maintenance of cell homeostasis (Updegrove et al 2015; Wagner and Romby 2015), and the modulation of bacterial virulence (Ellis et al 2017; Heidrich et al 2017). Magnesium ion homeostasis in bacterial cells is regulated by the PhoP/PhoQ two-component system (Groisman et al 2013). This system controls numerous genes involved in Mg2+ uptake (Wang et al 2017) and cell surface modification (Moon et al 2013). MgrR is a PhoP/PhoQ-dependent sRNA, and its transcription is induced in response to moderately low Mg2+ and Ca2+ levels (Moon and Gottesman 2009). MgrR was proposed to regulate numerous mRNA targets, but it had the largest effects on the levels of eptB mRNA, which encodes a lipopolysaccharide modification enzyme, and ygdQ mRNA, which encodes an inner membrane protein of unknown function (Moon and Gottesman 2009; Moon et al 2013; Lee and Gottesman 2016; Melamed et al 2016)

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