Mutations in Interaction Surfaces Differentially Impact E. coli Hfq Association with Small RNAs and Their mRNA Targets

Abstract

The RNA chaperone protein Hfq is required for the function of all small RNAs (sRNAs) that regulate mRNA stability or translation by limited base pairing in E. coli. While there have been numerous in vitro studies to characterize Hfq activity and the importance of specific residues, there has been only limited characterization of Hfq mutants in vivo. Here we use a set of reporters as well as co-immunoprecipitation to examine 14 Hfq mutants expressed from the E. coli chromosome. The majority of the proximal face residues, as expected, were important for the function of sRNAs. The failure of sRNAs to regulate target mRNAs in these mutants can be explained by reduced sRNA accumulation. Two of the proximal mutants, D9A and F39A, acted differently from the others in that they had mixed effects on different sRNA/mRNA pairs and, in the case of F39A, showed differential sRNA accumulation. Mutations of charged residues at the rim of Hfq interfered with positive regulation, and gave mixed effects for negative regulation. Some, but not all, sRNAs accumulated to lower levels in rim mutants, suggesting qualitative differences in how individual sRNAs are affected by Hfq. The distal face mutants were expected to disrupt binding of ARN motifs found in mRNAs. They were more defective for positive regulation than negative regulation at low mRNA expression, but the defects could be suppressed by higher levels of mRNA expression. We discuss the implications of these observations for Hfq binding to RNA and mechanisms of action.

Clostridium difficile Hfq can replace Escherichia coli Hfq for most of its function

“…These amino acids have defined three regions in the Hfq structure, two important for the interaction with sRNAs, the proximal face and lateral surface, while the proximal face is thought to contact the mRNA (Mikulecky et al 2004;Ziolkowska et al 2006;Sauer et al 2012;Zhang et al 2013). As described above, the Hfq protein of C. difficile retains most but not all the critical residues found to be essential for the function of the E. coli protein.…”

“…A number of amino acids in the E. coli protein have been identified as important for interaction with RNA. Three RNA-binding sites were defined by groups of residues and called the proximal (Q8, D9, F39, Y55, K56, F42), distal (Y25, I30), and the rim (R16, R19, and R17) RNA-binding surfaces (Mikulecky et al 2004;Zhang et al 2013). While the core of the various Hfq paralogs is rather conserved in sequence and structure (residues 7-66 in E. coli) (Fig.…”

“…The ortholog cluster multiple alignment of the amino acids sequences of Hfq proteins of several Clostridia and various Gram-positive or Gram-negative model species was performed by using ClustalW on the website http://mbgd.genome.ad.jp. Proximal face residues important for the interaction with RNA are highlighted in gray, those important for distal face interaction and at the rim are double underlined and underlined, respectively, both in the Ec-Hfq (Mikulecky et al 2004;Zhang et al 2013) and Cd-Hfq. The numbering is based on Ec-Hfq.…”

“…proximal binding surface (Q8, D9, F39, Y55, K56, but not F42 which is replaced by Y, also an aromatic residue), in the distal site (Y25, I30) and at the rim (R16, R19, but not R17, replaced by K and which retains the positive charge) (Mikulecky et al 2004;Zhang et al 2013). However, the Cd-Hfq C-terminal domain is very different; it is much shorter (16 aa) and includes an unusual stretch of seven asparagine residues, unique to this species (Fig.…”

Duplex formation between the sRNA DsrA andrpoSmRNA is not sufficient for efficient RpoS synthesis at low temperature

“…The RNA chaperone Hfq has been shown to be crucial for riboregulation in Enterobacteriaceae, which results on the one hand from binding to and protection of sRNAs from nucleolytic decay, [3][4][5] and on the other hand from accelerating annealing of sRNAs with their target mRNAs. 3 E. coli Hfq hexamers have dedicated RNA binding sites, preferably binding uridine-rich stretches of sRNAs around the central pore of the proximal surface [6][7][8][9][10] and A-rich sequences on the distal surface. 11 Moreover, binding of the sRNA RybB to Hfq not only involves contacts via 3′ terminal U-rich stretches but also with the lateral surface of the hexamer.…”