The RNA chaperone Hfq plays crucial roles in bacterial gene expression and is a major facilitator of small regulatory RNA (sRNA) action. The toroidal architecture of the Hfq hexamer presents three well-characterized surfaces that allow it to bind sRNAs to stabilize them and engage target transcripts. Hfq-interacting sRNAs are categorized into two classes based on the surfaces they use to bind Hfq. By characterizing a systematic alanine mutant library of Hfq to identify amino acid residues that impact survival of Escherichia coli experiencing nitrogen (N) starvation, we corroborated the important role of the three RNA-binding surfaces for Hfq function. We uncovered two, previously uncharacterized, conserved residues, V22 and G34, in the hydrophobic core of Hfq, to have a profound impact on Hfq's RNA-binding activity in vivo. Transcriptome-scale analysis revealed that V22A and G34A Hfq mutants cause widespread destabilization of both sRNA classes, to the same extent as seen in bacteria devoid of Hfq. However, the alanine substitutions at these residues resulted in only modest alteration in stability and structure of Hfq. We propose that V22 and G34 have impact on Hfq function, especially critical under cellular conditions when there is an increased demand for Hfq, such as N starvation.

Centre for Bacterial Resistance Biology Imperial College London LondonSW7 2AZ United Kingdom

Core Unit Systems Medicine University of Würzburg D 97080 Würzburg Germany

Department of Biochemistry University of Cambridge CambridgeCB2 1GA United Kingdom

Institute of Biophysics of the Czech Academy of Sciences Kralovopolska 135 Brno612 00 Czech Republic

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Vogel  J, Luisi  BF.  Hfq and its constellation of RNA. Nat Rev Microbiol. 2011; 9:578–89. PubMed PMC

Melamed  S, Adams  PP, Zhang  A  et al. .  RNA–RNA interactomes of ProQ and Hfq reveal overlapping and competing roles. Mol Cell. 2020; 77:411–25. PubMed PMC

Urban  JH, Vogel  J.  Two seemingly homologous noncoding RNAs act hierarchically to activate glmS mRNA translation. PLoS Biol. 2008; 6:e64. PubMed PMC

Vytvytska  O, Moll  I, Kaberdin  VR  et al. .  Hfq (HF1) stimulates ompA mRNA decay by interfering with ribosome binding. Genes Dev. 2000; 14:1109–18. PubMed PMC

Pei  XY, Dendooven  T, Sonnleitner  E  et al. .  Architectural principles for Hfq/Crc-mediated regulation of gene expression. eLife. 2019; 8:e43158. PubMed PMC

Hajnsdorf  E, Regnier  P.  Host factor Hfq of Escherichia coli stimulates elongation of poly(A) tails by poly(A) polymerase I. Proc Natl Acad Sci USA. 2000; 97:1501–5. PubMed PMC

Mohanty  BK, Maples  VF, Kushner  SR.  The Sm-like protein Hfq regulates polyadenylation dependent mRNA decay in Escherichia coli. Mol Microbiol. 2004; 54:905–20. PubMed

Wilusz  CJ, Wilusz  J.  Eukaryotic Lsm proteins: lessons from bacteria. Nat Struct Mol Biol. 2005; 12:1031–36. PubMed

Kambach  C, Walke  S, Young  R  et al. .  Crystal structures of two Sm protein complexes and their implications for the assembly of the spliceosomal snRNPs. Cell. 1999; 96:375–87. PubMed

Orans  J, Kovach  AR, Hoff  KE  et al. .  Crystal structure of an Escherichia coli Hfq core (residues 2–69)–DNA complex reveals multifunctional nucleic acid binding sites. Nucleic Acids Res. 2020; 48:3987–97. PubMed PMC

Horstmann  N, Orans  J, Valentin-Hansen  P  et al. .  Structural mechanism of Staphylococcus aureus Hfq binding to an RNA A-tract. Nucleic Acids Res. 2012; 40:11023–35. PubMed PMC

Link  TM, Valentin-Hansen  P, Brennan  RG.  Structure of Escherichia coli Hfq bound to polyriboadenylate RNA. Proc Natl Acad Sci USA. 2009; 106:19292–7. PubMed PMC

Santiago-Frangos  A, Frohlich  KS, Jeliazkov  JR  et al. .  Caulobacter crescentus Hfq structure reveals a conserved mechanism of RNA annealing regulation. Proc Natl Acad Sci USA. 2019; 116:10978–87. PubMed PMC

Sauer  E, Schmidt  S, Weichenrieder  O.  Small RNA binding to the lateral surface of Hfq hexamers and structural rearrangements upon mRNA target recognition. Proc Natl Acad Sci USA. 2012; 109:9396–401. PubMed PMC

Santiago-Frangos  A, Kavita  K, Schu  DJ  et al. .  C-terminal domain of the RNA chaperone Hfq drives sRNA competition and release of target RNA. Proc Natl Acad Sci USA. 2016; 113:E6089–96. PubMed PMC

Santiago-Frangos  A, Jeliazkov  JR, Gray  JJ  et al. .  Acidic C-terminal domains autoregulate the RNA chaperone Hfq. eLife. 2017; 6:e27049. PubMed PMC

Kavita  K, Zhang  A, Tai  CH  et al. .  Multiple in vivo roles for the C-terminal domain of the RNA chaperone Hfq. Nucleic Acids Res. 2022; 50:1718–33. PubMed PMC

Schu  DJ, Zhang  A, Gottesman  S  et al. .  Alternative Hfq–sRNA interaction modes dictate alternative mRNA recognition. EMBO J. 2015; 34:2557–73. PubMed PMC

Malecka  EM, Woodson  SA.  RNA compaction and iterative scanning for small RNA targets by the Hfq chaperone. Nat Commun. 2024; 15:2069. PubMed PMC

McQuail  J, Matera  G, Grafenhan  T  et al. .  Global Hfq-mediated RNA interactome of nitrogen starved Escherichia coli uncovers a conserved post-transcriptional regulatory axis required for optimal growth recovery. Nucleic Acids Res. 2024; 52:2323–39. PubMed PMC

McQuail  J, Switzer  A, Burchell  L  et al. .  The RNA-binding protein Hfq assembles into foci-like structures in nitrogen starved Escherichia coli. J Biol Chem. 2020; 295:12355–67. PubMed PMC

Walling  LR, Kouse  AB, Shabalina  SA  et al. .  A 3' UTR-derived small RNA connecting nitrogen and carbon metabolism in enteric bacteria. Nucleic Acids Res. 2022; 50:10093–109. PubMed PMC

Switzer  A, Burchell  L, McQuail  J  et al. .  The adaptive response to long-term nitrogen starvation in Escherichia coli requires the breakdown of allantoin. J Bacteriol. 2020; 202:e00172-20. PubMed PMC

Atlas  RM.  Handbook of Microbiological Media. 2010; 4th ednLouisville, KY, USA: CRC Press.

Sauter  C, Basquin  Jrm, Suck  D.  Sm-like proteins in eubacteria: the crystal structure of the Hfq protein from Escherichia coli. Nucleic Acids Res. 2003; 31:4091–98. PubMed PMC

Case  DA, Aktulga  HM, Belfon  K  et al. .  AmberTools24. 2021; San Francisco, CA, USA: University of California.

Maier  JA, Martinez  C, Kasavajhala  K  et al. .  ff14SB: improving the accuracy of protein side chain and backbone parameters from ff99SB. J Chem Theory Comput. 2015; 11:3696–713. PubMed PMC

Krepl  M, Pokorná  P, Mlýnský  V  et al. .  Spontaneous binding of single-stranded RNAs to RRM proteins visualized by unbiased atomistic simulations with a rescaled RNA force field. Nucleic Acids Res. 2022; 50:12480–96. PubMed PMC

Tian  C, Kasavajhala  K, Belfon  KAA  et al. .  ff19SB: amino-acid-specific protein backbone parameters trained against quantum mechanics energy surfaces in solution. J Chem Theory Comput. 2020; 16:528–52. PubMed

Izadi  S, Anandakrishnan  R, Onufriev  AV.  Building water models: a different approach. J Phys Chem Lett. 2014; 5:3863–71. PubMed PMC

Berendsen  HJC, Grigera  JR, Straatsma  TP.  The missing term in effective pair potentials. J Phys Chem. 1987; 91:6269–71.

Joung  IS, Cheatham  TE.  Determination of alkali and halide monovalent ion parameters for use in explicitly solvated biomolecular simulations. J Phys Chem B. 2008; 112:9020–41. PubMed PMC

Krepl  M, Vögele  J, Kruse  H  et al. .  An intricate balance of hydrogen bonding, ion atmosphere and dynamics facilitates a seamless uracil to cytosine substitution in the U-turn of the neomycin-sensing riboswitch. Nucleic Acids Res. 2018; 46:6528–43. PubMed PMC

Ryckaert  JP, Ciccotti  G, Berendsen  HJC.  Numerical integration of Cartesian equations of motion of a system with constraints—molecular dynamics of n-alkanes. J Comput Phys. 1977; 23:327–41.

Hopkins  CW, Le Grand  S, Walker  RC  et al. .  Long-time-step molecular dynamics through hydrogen mass repartitioning. J Chem Theory Comput. 2015; 11:1864–74. PubMed

Darden  T, York  D, Pedersen  L.  Particle mesh Ewald—an N.Log(N) method for Ewald sums in large systems. J Chem Phys. 1993; 98:10089–92.

Wang  L, Friesner  RA, Berne  BJ.  Replica exchange with solute scaling: a more efficient version of replica exchange with solute tempering (REST2). J Phys Chem B. 2011; 115:9431–38. PubMed PMC

Roe  DR, Cheatham  TE.  PTRAJ and CPPTRAJ: software for processing and analysis of molecular dynamics trajectory data. J Chem Theory Comput. 2013; 9:3084–95. PubMed

Humphrey  W, Dalke  A, Schulten  K.  VMD: visual molecular dynamics. J Mol Graph. 1996; 14:33–8. PubMed

Afonine  PV, Grosse-Kunstleve  RW, Echols  N  et al. .  Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr D Biol Crystallogr. 2012; 68:352–67. PubMed PMC

Murshudov  GN, Skubak  P, Lebedev  AA  et al. .  REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr D Biol Crystallogr. 2011; 67:355–67. PubMed PMC

Emsley  P, Lohkamp  B, Scott  WG  et al. .  Features and development of Coot. Acta Crystallogr D Biol Crystallogr. 2010; 66:486–501. PubMed PMC

Stead  MB, Agrawal  A, Bowden  KE  et al. .  RNAsnap: a rapid, quantitative and inexpensive, method for isolating total RNA from bacteria. Nucleic Acids Res. 2012; 40:e156. PubMed PMC

Martin  M.  Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J. 2011; 17:10–2.

Forstner  KU, Vogel  J, Sharma  CM.  READemption—a tool for the computational analysis of deep-sequencing-based transcriptome data. Bioinformatics. 2014; 30:3421–3. PubMed

Hoffmann  S, Otto  C, Kurtz  S  et al. .  Fast mapping of short sequences with mismatches, insertions and deletions using index structures. PLoS Comput Biol. 2009; 5:e1000502. PubMed PMC

Love  MI, Huber  W, Anders  S.  Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014; 15:550. PubMed PMC

Zhang  A, Schu  DJ, Tjaden  BC  et al. .  Mutations in interaction surfaces differentially impact E. coli Hfq association with small RNAs and their mRNA targets. J Mol Biol. 2013; 425:3678–97. PubMed PMC

Mikulecky  PJ, Kaw  MK, Brescia  CC  et al. .  Escherichia coli Hfq has distinct interaction surfaces for DsrA, rpoS and poly(A) RNAs. Nat Struct Mol Biol. 2004; 11:1206–14. PubMed PMC

Dimastrogiovanni  D, Frohlich  KS, Bandyra  KJ  et al. .  Recognition of the small regulatory RNA RydC by the bacterial Hfq protein. eLife. 2014; 3:e05375. PubMed PMC

Melamed  S, Peer  A, Faigenbaum-Romm  R  et al. .  Global mapping of small RNA–target interactions in bacteria. Mol Cell. 2016; 63:884–97. PubMed PMC

Fuchs  M, Lamm-Schmidt  V, Lence  T  et al. .  A network of small RNAs regulates sporulation initiation in Clostridioides difficile. EMBO J. 2023; 42:e112858. PubMed PMC

Huber  M, Lippegaus  A, Melamed  S  et al. .  An RNA sponge controls quorum sensing dynamics and biofilm formation in Vibrio cholerae. Nat Commun. 2022; 13:7585. PubMed PMC

Matera  G, Altuvia  Y, Gerovac  M  et al. .  Global RNA interactome of Salmonella discovers a 5′ UTR sponge for the MicF small RNA that connects membrane permeability to transport capacity. Mol Cell. 2022; 82:629–44. PubMed

Pearl Mizrahi  S, Elbaz  N, Argaman  L  et al. .  The impact of Hfq-mediated sRNA–mRNA interactome on the virulence of enteropathogenic Escherichia coli. Sci Adv. 2021; 7:eabi8228. PubMed PMC

Schumacher  MA, Pearson  RF, Moller  T  et al. .  Structures of the pleiotropic translational regulator Hfq and an Hfq–RNA complex: a bacterial Sm-like protein. EMBO J. 2002; 21:3546–56. PubMed PMC

Robinson  KE, Orans  J, Kovach  AR  et al. .  Mapping Hfq–RNA interaction surfaces using tryptophan fluorescence quenching. Nucleic Acids Res. 2014; 42:2736–49. PubMed PMC

Kwiatkowska  J, Wroblewska  Z, Johnson  KA  et al. .  The binding of class II sRNA MgrR to two different sites on matchmaker protein Hfq enables efficient competition for Hfq and annealing to regulated mRNAs. RNA. 2018; 24:1761–84. PubMed PMC

Switzer  A, Evangelopoulos  D, Figueira  R  et al. .  A novel regulatory factor affecting the transcription of methionine biosynthesis genes in Escherichia coli experiencing sustained nitrogen starvation. Microbiology (Reading). 2018; 164:1457–70. PubMed

Andrade  JM, Dos Santos  RF, Chelysheva  I  et al. .  The RNA-binding protein Hfq is important for ribosome biogenesis and affects translation fidelity. EMBO J. 2018; 37:e97631. PubMed PMC

Lee  T, Feig  AL.  The RNA binding protein Hfq interacts specifically with tRNAs. RNA. 2008; 14:514–23. PubMed PMC

Bandyra  KJ, Bouvier  M, Carpousis  AJ  et al. .  The social fabric of the RNA degradosome. Biochim. Biophys. Acta. 2013; 1829:514–22. PubMed PMC

Luo  X, Zhang  A, Tai  CH  et al. .  An acetyltranferase moonlights as a regulator of the RNA binding repertoire of the RNA chaperone Hfq in Escherichia coli. Proc Natl Acad Sci USA. 2023; 120:e2311509120. PubMed PMC