The Role of Sm-Like Protein Hfq in RNA Annealing

Abstract

Small RNAs (sRNAs) regulate bacterial response and adaptation to environmental stress. This RNA regulation mechanism requires the Sm-like binding protein, Hfq, which promotes annealing of complimentary RNAs. The distal face of Hfq interacts with poly(A) sequences, while the proximal face interacts with poly(U) sequences. The sRNA DsrA binds to the rpoS mRNA, opening an inhibitory stem loop in the mRNA, and consequently initiating translation of the RpoS sigma stress response factor in Escherichia coli. Hfq binding to an (AAN)4 repeat element upstream of the inhibitor stem loop on rpoS mRNA has been shown to be required to promote DsrA annealing. To further understand the role of Hfq in RNA annealing, we studied the formation of the ternary complex between Hfq, sRNA DsrA, and mRNA rpoS mRNA. Two mutations were chosen to disrupt RNA binding to either the proximal or distal face of the Hfq hexamer. Fluorescence Anisotropy experiments were used to study the binding affinity of Hfq mutants to poly(U) oligomers. Y25D, on the distal face of Hfq, showed only a two fold decrease in binding affinity; whereas K56A, on the proximal face, showed a 5-fold decrease. Native gel mobility shift assays showed that only the K56A mutant formed a ternary complex with DsrA and rpoS RNAs. In contrast, the Y25D mutation inhibits rpoS mRNA binding and also fails to form a ternary complex. These results support previous results showing that Hfq binding to A-rich sequences in rpoS mRNA is critical for rpoS regulation.