Abstract
The RNA-chaperone Hfq catalyses the annealing of bacterial small RNAs (sRNAs) with target mRNAs to regulate gene expression in response to environmental stimuli. Hfq acts on a diverse set of sRNA-mRNA pairs using a variety of different molecular mechanisms. Here, we present an unusual crystal structure showing two Hfq-RNA complexes interacting via their bound RNA molecules. The structure contains two Hfq6:A18 RNA assemblies positioned face-to-face, with the RNA molecules turned towards each other and connected via interdigitating base stacking interactions at the center. Biochemical data further confirm the observed interaction, and indicate that RNA-mediated contacts occur between Hfq-RNA complexes with various (ARN)X motif containing RNA sequences in vitro, including the stress response regulator OxyS and its target, fhlA. A systematic computational survey also shows that phylogenetically conserved (ARN)X motifs are present in a subset of sRNAs, some of which share similar modular architectures. We hypothesise that Hfq can co-opt RNA-RNA base stacking, an unanticipated structural trick, to promote the interaction of (ARN)X motif containing sRNAs with target mRNAs on a “speed-dating” fashion, thereby supporting their regulatory function.
Highlights
Non-coding RNAs play key roles in regulating gene expression in all domains of life
Genomic SELEX experiments revealed a specific enrichment of A-rich sequences among Hfq-bound RNAs and in vivo UV-crosslinking demonstrated that Hfq binds to repeated ARN triplets (referred to as (ARN)X motifs) in the 5′-untranslated regions (UTR) of mRNAs34, 37
The crystals resulted from an experiment aimed at co-crystallizing Escherichia coli Hfq[72] with A30 RNA and poly(A)-polymerase 1, but they contain only Hfq[72] and an 18 nucleotide long poly(A) RNA segment
Summary
Non-coding RNAs play key roles in regulating gene expression in all domains of life. In bacteria, sRNAs control almost every aspect of bacterial physiology including metabolism, quorum sensing, and virulence[1,2,3,4]. The ‘proximal’ site on one face of the ring preferentially binds to U-rich RNA sequences, such as the poly(U) tracts present at the 3′ termini of most sRNAs32, 33 At these poly(U) tails, Hfq directly interacts with the free 3′-OH group, which helps trigger a constricted RNA conformation required for efficient sRNA binding and recognition[32]. Crystal structures revealed that the distal site of each Hfq subunit can accommodate a triplet of RNA nucleotides (ARN or AAN) with differing specificities: the A-site binds adenines, the R-site can accommodate both adenine and guanine with preference for A, while the third base points away from Hfq towards the solvent and can be any nucleotide (N)[26, 35] Six such sites come together in the hexamer to form a circular binding site accommodating an 18nt long A-rich RNA segment[36]. In addition to proximal site binding regions[39, 43], OxyS contains an extended (ARN)X motif (positions 59–86) that is essential for its regulatory function, and biochemical studies and crystal structures have shown that it binds to Hfq’s distal site[42, 46]
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