Hfq and its constellation of RNA

Abstract

Hfq is an RNA-binding protein that is common to diverse bacterial lineages and has key roles in the control of gene expression. By facilitating the pairing of small RNAs with their target mRNAs, Hfq affects the translation and turnover rates of specific transcripts and contributes to complex post-transcriptional networks. These functions of Hfq can be attributed to its ring-like oligomeric architecture, which presents two non-equivalent binding surfaces that are capable of multiple interactions with RNA molecules. Distant homologues of Hfq occur in archaea and eukaryotes, reflecting an ancient origin for the protein family and hinting at shared functions. In this Review, we describe the salient structural and functional features of Hfq and discuss possible mechanisms by which this protein can promote RNA interactions to catalyse specific and rapid regulatory responses in vivo.

Figure 1. Widely accepted modes of Hfq activity

a ∣ Hfq in association with a small RNA (sRNA) may sequester the ribosome-binding site (RBS) of a target mRNA, thus blocking binding of the 30S and 50S ribosomal subunits and repressing translation. b ∣ In some mRNAs, a secondary structure in the 5′ untranslated region (UTR) can mask the RBS and inhibit translation. A complex formed by Hfq and a specific sRNA may activate the translation of one of these mRNAs by exposing the translation initiation region for 30S binding^,. c ∣ Hfq may protect some sRNAs from ribonuclease cleavage, which is carried out by ribonuclease E (RNase E) in many cases. d ∣ Hfq may induce the cleavage (often by RNase E^,,) of some sRNAs and their target mRNAs. e ∣ Hfq may stimulate the polyadenylation of an mRNA by poly(A) polymerase (PAP), which in turn triggers 3′-to-5′ degradation by an exoribonuclease (Exo)^,. In Escherichia coli, the exoribonuclease can be polynucleotide phosphorylase, RNase R or RNase II.

Figure 2. The structure of Hfq and its interactions with RNA

a ∣ Secondary-structural elements of the Hfq protomer, highlighting the conserved Sm1 and Sm2 sequence motifs. b,c ∣ Each protomer is a compact α–β_1–5 structural unit (that is, composed of one α-helix and five β-strands) in which the β-strands form a set of antiparallel sheets. One of the strands (β₂) is twisted and curved to such an extent that it contributes to both sheets to form a self-closing, squat barrel (part c). The amino-terminal helix and squat barrel are structural signatures of Hfq–Sm–Sm-like (LSm) proteins and groups them into the wider oligonucleotide–oligosaccharide (OB)-fold structural class, members of which include the highly conserved single-stranded DNA-binding proteins^,. The β₄ and β₅ strands on the periphery of each Hfq subunit expose hydrogen-bonding edges that interact with the strands of the neighbouring protomers, so that sheets effectively continue over the entire ring. The organization of secondary-structural elements and protomer–protomer contacts is similar in the hetero-heptameric Sm assembly of the human spliceosome (not shown). The inter-strand angles define the spatial relationship of the protomers and, consequently, the number of subunits within the ring. d ∣ Two faces for interaction with RNAs (orange) are presented on opposite sides of the Hfq ring. The proximal face (the surface on which the amino-terminal α-helix is exposed) includes residues in the Sm2 sequence motif. Disordered tails are likely to emanate from the equator of the Hfq ring and may form distributive electrostatic interactions with nucleic acids.

Figure 3. A typical Hfq-associated small RNA

a ∣ The domain structure of RybB, a small RNA (sRNA) from Salmonella enterica^,, showing the location of the Hfq-binding region and a potential site of Hfq interaction with the 3′ poly(U) tail^,. The ‘seed’ region of the sRNA, for mRNA recognition, is shown in an orange box, and the predicted secondary structure of the transcription terminator is the hairpin structure on the right. Hfq also protects regions in the terminator structure from attack by enzymes and chemical probes, suggesting that additional interactions may exist between Hfq and the sRNA. b ∣ A comparison of the dimensions of Hfq and RybB sRNA (according to the in silico predicted structure of RybB). This provides an impression of scale as an indication of the potential extent of interaction between Hfq and RNAs.

Figure 4. A three-body problem involving Hfq, small RNA and mRNA

a ∣ Different scenarios envisaged for resolution of the ternary complex formed between Hfq, the small RNA (sRNA) and the target mRNA. An sRNA–mRNA pairing could be the final product (left), the mRNA–Hfq complex could be the most stable product of the encounter (middle), or the ternary complex could bind to other proteins (right; ribonuclease E (RNase E) is shown as a representative effector protein). The schematic is deliberately ambiguous about which face of the Hfq hexamer the RNAs are binding. b ∣ A highly speculative energy landscape for the three-body problem. The graph shows the catalytic role of Hfq. Hfq accelerates the association rate (or on rate), depicted as decreasing the activation barrier for complex formation. It also stabilizes the equilibrium duplex structure of sRNA–mRNA, shown on the right of the reaction coordinate, perhaps by driving metastable structures to a local energy minimum.

Figure 5. A model for RNA exchange on Hfq

The arrangement of multiple RNA-binding sites enables the piecemeal displacement of tightly bound RNA by free RNA. This model accounts for the rapid responses of small RNA (sRNA) function in vivo, but it raises the question of how specificity arises in the face of competition from abundant cellular RNAs. Another problem is that sRNAs are not stable in the absence of Hfq, so a large concentration burst of a competing RNA would be required in vivo to favour exchange. Figure is modified, with permission, from REF. 38 © (2010) Cold Spring Harbor Laboratory Press.