Structure and RNA-binding properties of the bacterial LSm protein Hfq

Abstract

Over the past years, small non-coding RNAs (sRNAs) emerged as important modulators of gene expression in bacteria. Guided by partial sequence complementarity, these sRNAs interact with target mRNAs and eventually affect transcript stability and translation. The physiological function of sRNAs depends on the protein Hfq, which binds sRNAs in the cell and promotes the interaction with their mRNA targets. This important physiological function of Hfq as a central hub of sRNA-mediated regulation made it one of the most intensely studied proteins in bacteria. Recently, a new model for sRNA binding by Hfq has been proposed that involves the direct recognition of the sRNA 3' end and interactions of the sRNA body with the lateral RNA-binding surface of Hfq. This review summarizes the current understanding of the RNA binding properties of Hfq and its (s)RNA complexes. Moreover, the implications of the new binding model for sRNA-mediated regulation are discussed.

Keywords: 3′ end recognition; LSm ring; RNA chaperone; RNA degradation; crystal structure; gene regulation; non-coding RNAs; prokaryotes.

Figure 1. Fold and oligomerization of the LSm domain. (A) Cartoon representation of the LSm domain of Salmonella typhimurium Hfq (PDB-ID: 2YLB⁹). Secondary structure elements (α-helix 1, red; β-sheets 1–5, green; loops 1–4, white) as well as the N- and C-termini are indicated. The five β-strands form a half-open barrel with the N-terminal α-helix stacked on top. (B) Cartoon representation of the Salmonella typhimurium Hfq₆ ring. A single LSm domain is highlighted and colored as in (A); secondary structure elements involved in intersubunit interactions are colored green. Six LSm domains assemble into a homohexameric ring resulting in an extended β-sheet spanning the entire hexamer. Intersubunit contacts are provided by backbone interactions between strands β4 and β5 to strands β5* and β4* in the neighboring (indicated by a *) monomers, respectively. All (L)Sm rings assemble in a polar way with the N-terminal α-helices located on the same side of the oligomer. (C) Multiple sequence alignment of bacterial Hfq proteins. The secondary structure of Salmonella typhimurium Hfq (PDB-ID: 2YLB⁹) is superimposed on the primary sequence. The Sm consensus sequences are shown below the alignment (the nature of the amino acid side-chains is: s = small hydrophobic, I, L, V; h = hydrophilic, S, T; a = aromatic, Y, F). Highly conserved residues are red (> 70% conservation) or white in red boxes (100% conservation). While the Sm1 signature is conserved in all domains of life, the Sm2 is divergent in bacteria. The species abbreviations and UniProt-IDs are: γ-Proteobacteria: SALTY, Salmonella typhimurium (P0A1R0); ECOLI Escherichia coli (P0A6X3); YERPE, Yersinia pestis (A4TRN9); HAEIN, Hemophilus influenza, (P44437); LEGPA Legionella pneumophila (Q5X982). β-Proteobacteria: NEIME, Neisseiriameningitides (B9VV05); RALSO, Ralstonia solanacearum (Q8Y025). α-Proteobacteria: GLUDI, Gluconacetobacter diazotrophicus (Q8RMG6); Acidobacteria: ACIBL, Acidobacteria bacterium (Q1IIF9). Spirochaetales: LEPIN, Leptospira interogans (Q8F5Z7). Aquafecales: AQUAE Aquifex aeolicus, (O66512). Thermotogales: THEMA, Thermotoga maritime (Q9WYZ6). Fermicutes: BACSU, Bacillus subtilis (O31796); STAAM, Staphylococcus aureus (Q99UG9).

Figure 2. The LSm fold is conserved in all domains of life. Multiple sequence alignment of the LSm domain found in bacterial (Hfq), archeal (aLSM) and human (L)Sm proteins. Residues with > 70% sequence conservation are shown in red. The secondary structure of always the top species of each subgroup is indicated on top of the primary sequence. Although the sequence conservation of the different LSm domains is low, the LSm fold is conserved. The main differences are the length of the N-terminal α-helix, the strands β3 and β4 as well as loop L1 and L4. Interestingly, the only known archeal homohexameric Hfq protein (aLSM_METJA) comprises an Sm2 signature very similar to its homohexameric bacterial homologs, while the Sm2 motif of homoheptameric archeal homologs is more related to the human (L)Sm proteins. Species abbreviations and UniProt accession numbers for the selected bacterial sequences are given in Figure 1. Species abbreviations and UniProt accession numbers of archeal LSm proteins are: Methanococci: METJA, Methanococcus jannaschii (Q58830); Archeoglobi: ARCFU, Archeoglobus fulgidus, (O29386); Thermoplasmata: THEAC, Thermoplasma acidophilum, (P57670); Methanomicrobia: METMJ, Methanoculleus marisnigri, (A3CS14); Methanobacteria: METTH, Methanobacterium thermoautotrophicum, (O26745); Thermococci: PYRAB, Pyrococcus abyssi, (Q9V0Y8); Methanopyri: METKA, Methanopyrus kandleri, (Q8TYS2); Methanococci: METMP, Methanococcus maripaludis, (Q6LY45); Halobacteria: HALSA, Halobacterium salinarum, (Q9HPS2); Nanoarcheoata NANEQ, Nanoarcheum equitans, (Q74N54); Thaumarcheoata CENSY, Cenarcheum symbiosum, (A0RZA4); Crenarcheoata SULSO, Sulfolubus solfataricus, (Q97ZQ0); Crenarcheoata PYRAE, Pyrobacculum aerophilum, (Q8ZYG5); Koracheoata CORCO, Korarcheum cryptofilum, (B1L734). The accession numbers for the HUMAN (Homo sapiens) Sm proteins are: SmD1, (P62314); SmD3, (P62318); SmE, (P62304); SmF, (P62306); SmG, (P62308). LSm proteins: LSm1, (O15116); LSm2, (Q9Y333); LSm4, (Q9Y4Z0); LSm5, (Q9Y4Y9); LSm6, (P62312); LSm8, (O95777).

Figure 3. Model of an Hfq/sRNA complex. Proximal side view of a model of the Hfq/RybB complex. The Hfq hexamer (PDB-ID: 2YLC⁹) is shown as a surface representation with a superimposed model of only one of the six Hfq C-termini for clarity. RybB sRNA is depicted in cartoon representation with the ρ-independent terminator colored in green, the single-stranded sequence is blue and the location of the seed region is indicated. The asterisks mark the location of the six lateral RNA binding sites of Hfq. The model was assembled using COOT considering the biochemical and structural evidence summarized in this review. The depicted structure of the C terminus was modeled using HHpred. The model shows how Hfq might interact with RybB sRNA and also gives an impression of the proportions of the sRNA body with respect to the size of the terminator stem-loop and the Hfq protein.