Acidic Residues in the Hfq Chaperone Increase the Selectivity of sRNA Binding and Annealing

Abstract

Hfq facilitates gene regulation by small non-coding RNAs (sRNAs), thereby affecting bacterial attributes such as biofilm formation and virulence. E. coli Hfq recognizes specific U-rich and AAN motifs in sRNAs and target mRNAs, after which an arginine patch on the rim promotes base pairing between their complementary sequences. In the cell, Hfq must discriminate between many similar RNAs. Here, we report that acidic amino acids lining the sRNA binding channel between the inner pore and rim of the Hfq hexamer contribute to the selectivity of Hfq’s chaperone activity. RNase footprinting, in vitro binding and stopped-flow fluorescence annealing assays showed that alanine substitution of D9, E18 or E37 strengthened RNA interactions with the rim of Hfq and increased annealing of non-specific or U-tailed RNA oligomers. Although the mutants were less able than WT Hfq to anneal sRNAs with wild type rpoS mRNA, the D9A mutation bypassed recruitment of Hfq to an (AAN)4 motif in rpoS, both in vitro and in vivo. These results suggest that acidic residues normally modulate access of RNAs to the arginine patch. We propose that this selectivity limits indiscriminate target selection by E. coli Hfq, and enforces binding modes that favor genuine sRNA and mRNA pairs.

Arginine Patch Predicts the RNA Annealing Activity of Hfq from Gram-Negative and Gram-Positive Bacteria

“…2b, c). As previously observed (19), the rate of RNA annealing increased with added E. coli Hfq, reaching a maximum value in 50 nM Hfq 6 , which equals the amount of molecular beacon. For target-U6 RNA, annealing was 15 times faster with 50 nM Hfq, compared to the no Hfq background (0.08 s −1 to 1.3 s −1 ; Fig.…”

“…Importantly, complementary sequences in the sRNA and mRNA target also interact with an arginine patch (R16, R17 and R19) on the rim of the E. coli Hfq hexamer (13, 14, 15, 16, 17) that is essential for Hfq’s RNA annealing activity (15). In vitro annealing assays showed that E. coli Hfq accelerates sRNA-mRNA base pairing 30 to 100 times by nucleating the double helix between two complementary RNA strands (18, 19). This annealing activity is eliminated when all three arginines are replaced with alanine, and even an arginine to lysine substitution reduces E. coli Hfq’s chaperone activity (15).…”

C-terminal domain of the RNA chaperone Hfq drives sRNA competition and release of target RNA

“…Although fulllength Hfq102 prioritizes Class II sRNAs, Hfq65 binds most sRNAs equally. Because Hfq is limiting in the cell (46), this redistribution of Hfq could disrupt sRNA-mRNA regulatory networks or affect the accumulation of sRNAs when the CTD is absent (31).…”

Hfq: the flexible RNA matchmaker

“…Nonetheless, a study that examined sRNA accumulation and target mRNA regulation in E. coli strains expressing various Hfq mutants from the chromosome [16], showed that while mutations in some conserved proximal face residues (such as Q8, F42 and K56) affected most sRNAs analyzed, mutations in other proximal face residues (such as D9 and F39) had varying effects toward different sRNAs. It also was recently reported that certain acidic residues (D9, E18 and E37) on the proximal face of the E. coli protein are important for the discrimination of sRNAs from other cellular RNAs; mutations of these residues allowed for more non-discriminant Hfq binding to RNA while reducing the efficiency of the sRNA-mRNA annealing [17]. Generally, although sRNAs appear to be similarly anchored to the proximal face of Hfq via the polyU tail of the Rho-independent terminator, there are some differences in the way individual sRNAs contact this face.…”