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Escherichia coli Hfq has distinct interaction surfaces for DsrA, rpoS and poly(A) RNAs

Nature Structural & Molecular Biology volume 11, pages 1206–1214 (2004)Cite this article

Abstract

The bacterial Sm-like protein Hfq facilitates RNA-RNA interactions involved in post-transcriptional regulation of the stress response. Specifically, Hfq helps pair noncoding RNAs (ncRNAs) with complementary regions of target mRNAs. To probe the mechanism of this pairing, we generated a series of Hfq mutants and measured their affinity for RNAs like those with which Hfq must associate in vivo. We tested the mutants' DsrA-dependent activation of rpoS, and their ability to stabilize DsrA ncRNA against degradation in vivo. Our results suggest that Hfq has two independent RNA-binding surfaces. In addition to a well-known site around the core of the Hfq hexamer, we observe interactions with the distal face of Hfq, a new locus with which mRNAs and poly(A) sequences associate. Our model explains how Hfq can simultaneously bind a ncRNA and its mRNA target to facilitate the strand displacement reaction required for Hfq-dependent translational regulation.

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Figure 1: Sequence alignments and structure of Hfq.
Figure 2: In vitro analysis of RNA binding to mutant Hfq proteins.
Figure 3: Native gel analysis showing the effect of A27 addition to the DsrA–(Hfq6)2 complex.
Figure 4: Assay for in vivo accumulation and rpoS activation.
Figure 5: Lifetime analysis of DsrA in vivo.

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References

  1. Hori, K. & Yanazaki, Y. Nucleotide sequence specific interaction of host factor I with bacteriophage Q β RNA. FEBS Lett. 43, 20–22 (1974).

    Article  CAS  PubMed  Google Scholar 

  2. Van Emmelo, J., De Boever, J., Gillis, E. & Fiers, W. A host factor required for Q-β-RNA-replication in “Escherichia coli”. Arch. Int. Physiol. Biochim. 81, 393–394 (1973).

    CAS  PubMed  Google Scholar 

  3. Storz, G. An Expanding Universe of Noncoding RNAs. Science 296, 1260–1263 (2002).

    Article  CAS  PubMed  Google Scholar 

  4. Repoila, F., Majdalani, N. & Gottesman, S. Small non-coding RNAs, co-ordinators of adaptation processes in Escherichia coli: the RpoS paradigm. Mol. Microbiol. 48, 855–861 (2003).

    Article  CAS  PubMed  Google Scholar 

  5. Storz, G., Opdyke, J.A. & Zhang, A. Controlling mRNA stability and translation with small, noncoding RNAs. Curr. Opin. Microbiol. 7, 140–144 (2004).

    Article  CAS  PubMed  Google Scholar 

  6. Valentin-Hansen, P., Eriksen, M. & Udesen, C. The bacterial Sm-like protein Hfq: a key player in RNA transactions. Mol. Microbiol. 51, 1525–1533 (2004).

    Article  CAS  PubMed  Google Scholar 

  7. Sledjeski, D.D., Whitman, C. & Zhang, A. Hfq is necessary for regulation by the untranslated RNA DsrA. J. Bacteriol. 183, 1997–2005 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Sonnleitner, E. et al. Reduced virulence of a hfq mutant of Pseudomonas aeruginosa O1. Microb. Pathog. 35, 217–228 (2003).

    Article  CAS  PubMed  Google Scholar 

  9. Majdalani, N., Cunning, C., Sledjeski, D., Elliott, T. & Gottesman, S. DsrA RNA regulates translation of RpoS message by an anti-antisense mechanism, independent of its action as an antisilencer of transcription. Proc. Natl. Acad. Sci. USA 95, 12462–12467 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Lease, R.A., Cusick, M.E. & Belfort, M. Riboregulation in Escherichia coli: DsrA RNA acts by RNA:RNA interactions at multiple loci. Proc. Natl. Acad. Sci. USA 95, 12456–12461 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Altuvia, S., Zhang, A., Argaman, L., Tiwari, A. & Storz, G. The Escherichia coli OxyS regulatory RNA represses fhlA translation by blocking ribosome binding. EMBO J. 17, 6069–6075 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Argaman, L. & Altuvia, S. fhlA repression by OxyS RNA: kissing complex formation at two sites results in a stable antisense–target RNA complex. J. Mol. Biol. 300, 1101–1112 (2000).

    Article  CAS  PubMed  Google Scholar 

  13. Zhang, A. et al. The OxyS regulatory RNA represses rpoS translation and binds the Hfq (HF-I) protein. EMBO J. 17, 6061–6068 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Majdalani, N., Chen, S., Murrow, J., St John, K. & Gottesman, S. Regulation of RpoS by a novel small RNA: the characterization of RprA. Mol. Microbiol. 39, 1382–1394 (2001).

    Article  CAS  PubMed  Google Scholar 

  15. Masse, E. & Gottesman, S. A small RNA regulates the expression of genes involved in iron metabolism in Escherichia coli . Proc. Natl. Acad. Sci. USA 99, 4620–4625 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Masse, E., Escorcia, F.E. & Gottesman, S. Coupled degradation of a small regulatory RNA and its mRNA targets in Escherichia coli . Genes Dev. 17, 2374–2383 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Vecerek, B., Moll, I., Afonyushkin, T., Kaberdin, V. & Blasi, U. Interaction of the RNA chaperone Hfq with mRNAs: direct and indirect roles of Hfq in iron metabolism of Escherichia coli . Mol. Microbiol. 50, 897–909 (2003).

    Article  CAS  PubMed  Google Scholar 

  18. Moller, T., Franch, T., Udesen, C., Gerdes, K. & Valentin-Hansen, P. Spot 42 RNA mediates discoordinate expression of the E. coli galactose operon. Genes Dev. 16, 1696–1706 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. 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 97, 1501–1505 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Le Derout, J. et al. Hfq affects the length and the frequency of short oligo(A) tails at the 3′ end of Escherichia coli rpsO mRNAs. Nucleic Acids Res. 31, 4017–4023 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Vytvytska, O., Moll, I., Kaberdin, V.R., von Gabain, A. & Blasi, U. Hfq (HF1) stimulates ompA mRNA decay by interfering with ribosome binding. Genes Dev. 14, 1109–1118 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Folichon, M. et al. The poly(A) binding protein Hfq protects RNA from RNase E and exoribonucleolytic degradation. Nucleic Acids Res. 31, 7302–7310 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Arluison, V. et al. Structural modelling of the Sm-like protein Hfq from Escherichia coli . J. Mol. Biol. 320, 705–712 (2002).

    Article  CAS  PubMed  Google Scholar 

  24. Moller, T. et al. Hfq: a bacterial Sm-like protein that mediates RNA–RNA interaction. Mol. Cell 9, 23–30 (2002).

    Article  CAS  PubMed  Google Scholar 

  25. Zhang, A., Wassarman, K.M., Ortega, J., Steven, A.C. & Storz, G. The Sm-like Hfq protein increases OxyS RNA interaction with target mRNAs. Mol. Cell 9, 11–22 (2002).

    Article  PubMed  Google Scholar 

  26. Sun, X., Zhulin, I. & Wartell, R.M. Predicted structure and phyletic distribution of the RNA-binding protein Hfq. Nucleic Acids Res. 30, 3662–3671 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Sauter, C., Basquin, J. & Suck, D. Sm-like proteins in eubacteria: the crystal structure of the Hfq protein from Escherichia coli . Nucleic Acids Res. 31, 4091–4098 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Marchler-Bauer, A. et al. CDD: a curated Entrez database of conserved domain alignments. Nucleic Acids Res. 31, 383–387 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Schumacher, M.A., Pearson, R.F., Moller, T., Valentin-Hansen, P. & Brennan, R.G. Structures of the pleiotropic translational regulator Hfq and an Hfq–RNA complex: a bacterial Sm-like protein. EMBO J. 21, 3546–3556 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Kambach, C. et al. Crystal structures of two Sm protein complexes and their implications for the assembly of the spliceosomal snRNPs. Cell 96, 375–387 (1999).

    Article  CAS  PubMed  Google Scholar 

  31. Achsel, T. et al. A doughnut-shaped heteromer of human Sm-like proteins binds to the 3′-end of U6 snRNA, thereby facilitating U4/U6 duplex formation in vitro . EMBO J. 18, 5789–5802 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Achsel, T., Stark, H. & Luhrmann, R. The Sm domain is an ancient RNA-binding motif with oligo(U) specificity. Proc. Natl. Acad. Sci. USA 98, 3685–3689 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Toro, I. et al. RNA binding in an Sm core domain: X-ray structure and functional analysis of an archaeal Sm protein complex. EMBO J. 20, 2293–2303 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Toro, I., Basquin, J., Teo-Dreher, H. & Suck, D. Archaeal Sm proteins form heptameric and hexameric complexes: crystal structures of the Sm1 and Sm2 proteins from the hyperthermophile Archaeoglobus fulgidus . J. Mol. Biol. 320, 129–142 (2002).

    Article  PubMed  Google Scholar 

  35. Moll, I., Leitsch, D., Steinhauser, T. & Blasi, U. RNA chaperone activity of the Sm-like Hfq protein. EMBO Rep. 4, 284–289 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Geissmann, T.A. & Touati, D. Hfq, a new chaperoning role: binding to messenger RNA determines access for small RNA regulator. EMBO J. 23, 396–405 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Brescia, C.C., Mikulecky, P.J., Feig, A.L. & Sledjeski, D.D. Identification of the Hfq binding site on DsrA RNA: Hfq binds without altering DsrA secondary structure. RNA 9, 33–43 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Moll, I., Afonyushkin, T., Vytvytska, O., Kaberdin, V.R. & Blasi, U. Coincident Hfq binding and RNase E cleavage sites on mRNA and small regulatory RNAs. RNA 9, 1308–1314 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Stage-Zimmermann, T.K. & Uhlenbeck, O.C. Hammerhead ribozyme kinetics. RNA 4, 875–889 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Plum, G.E. & Breslauer, K.J. Calorimetry of proteins and nucleic acids. Curr. Opin. Struct. Biol. 5, 682–690 (1995).

    Article  CAS  PubMed  Google Scholar 

  41. Ladbury, J.E. & Chowdhry, B.Z. Sensing the heat: the application of isothermal titration calorimetry to thermodynamic studies of biomolecular interactions. Chem. Biol. 3, 791–801 (1996).

    Article  CAS  PubMed  Google Scholar 

  42. Leavitt, S. & Freire, E. Direct measurement of protein binding energetics by isothermal titration calorimetry. Curr. Opin. Struct. Biol. 11, 560–566 (2001).

    Article  CAS  PubMed  Google Scholar 

  43. de Haseth, P.L. & Uhlenbeck, O.C. Interaction of Escherichia coli host factor protein with oligoriboadenylates. Biochemistry 19, 6138–6146 (1980).

    Article  CAS  PubMed  Google Scholar 

  44. Sledjeski, D.D., Gupta, A. & Gottesman, S. The small RNA, DsrA, is essential for the low temperature expression of RpoS during exponential growth in Escherichia coli . EMBO J. 15, 3993–4000 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Guzman, L.M., Belin, D., Carson, M.J. & Beckwith, J. Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J. Bacteriol. 177, 4121–4130 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Mura, C., Kozhukhovsky, A., Gingery, M., Phillips, M. & Eisenberg, D. The oligomerization and ligand-binding properties of Sm-like archaeal proteins (SmAPs). Protein Sci. 12, 832–847 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Thore, S., Mayer, C., Sauter, C., Weeks, S. & Suck, D. Crystal structures of the Pyrococcus abyssi Sm core and its complex with RNA: common features of RNA-binding in Archaea and Eukarya. J. Biol. Chem. 278, 1239–1247 (2003).

    Article  CAS  PubMed  Google Scholar 

  48. Urlaub, H., Raker, V.A., Kostka, S. & Luhrmann, R. Sm protein-Sm site RNA interactions within the inner ring of the spliceosomal snRNP core structure. EMBO J. 20, 187–196 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Stark, H., Dube, P., Luhrmann, R. & Kastner, B. Arrangement of RNA and proteins in the spliceosomal U1 small nuclear ribonucleoprotein particle. Nature 409, 539–542 (2001).

    Article  CAS  PubMed  Google Scholar 

  50. Arluison, V. et al. The C-terminal domain of Escherichia coli Hfq increases the stability of the hexamer. Eur. J. Biochem. 271, 1258–1265 (2004).

    Article  CAS  PubMed  Google Scholar 

  51. Stoscheck, C.M. Quantitation of protein. Methods Enzymol. 182, 50–68 (1990).

    Article  CAS  PubMed  Google Scholar 

  52. Mikulecky, P.J., Takach, J.C. & Feig, A.L. Entropy-driven folding of an RNA helical junction: an isothermal titration calorimetric analysis of the hammerhead ribozyme. Biochemistry 43, 5870–5881 (2004).

    Article  CAS  PubMed  Google Scholar 

  53. Schagger, H. & von Jagow, G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem. 166, 368–379 (1987).

    Article  CAS  PubMed  Google Scholar 

  54. Towbin, H., Staehelin, T. & Gordon, J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350–4354 (1979).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Brescia, C.C., Kaw, M.K. & Sledjeski, D.D. The DNA binding protein H-NS binds to and alters the stability of RNA in vitro and in vivo. J. Mol. Biol. 339, 505–514 (2004).

    Article  CAS  PubMed  Google Scholar 

  56. Bernstein, J.A., Khodursky, A.B., Lin, P.H., Lin-Chao, S. & Cohen, S.N. Global analysis of mRNA decay and abundance in Escherichia coli at single-gene resolution using two-color fluorescent DNA microarrays. Proc. Natl. Acad. Sci. USA 99, 9697–9702 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Miller, J.H. Experiments in Bacterial Genetics (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1972).

    Google Scholar 

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Acknowledgements

This work is supported by grants from the US National Institutes of Health (GM065430 to A.L.F., GM056448 to D.S., and T32-GM07757 to Indiana University/P.J.M.). A.L.F. is a Cottrell Scholar of Research Corporation. The Typhoon 9210 imaging system was purchased with a grant from the US National Science Foundation (DBI-0244815). The authors thank T. Stone (Indiana University) for technical support in the Physical Biochemistry Instrumentation Facility, and J. Fitzgerald, A. Kerzmann, N. Anderson and S. Kamel (Indiana University) for assistance in the cloning and sequencing of the constructs.

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Authors and Affiliations

  1. Department of Chemistry, Indiana University, 800 E. Kirkwood Avenue, Bloomington, 47405, Indiana, USA

    Peter J Mikulecky, Jennifer C Takach & Andrew L Feig

  2. Department of Microbiology and Immunology, Medical College of Ohio, 3055 Arlington Avenue, Toledo, 43614-5806, Ohio, USA

    Meenakshi K Kaw, Cristin C Brescia & Darren D Sledjeski

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  1. Peter J Mikulecky
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Correspondence to Andrew L Feig.

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Supplementary information

Supplementary Figure 1

Isothermal titration calorimetry of DsrA titrated into wild-type Hfq. (PDF 62 kb)

Supplementary Table 1

Summary of thermodynamic data from ITC analysis. (PDF 8 kb)

Supplementary Table 2

Primers used for cloning and QRT-PCR. (PDF 12 kb)

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Mikulecky, P., Kaw, M., Brescia, C. et al. Escherichia coli Hfq has distinct interaction surfaces for DsrA, rpoS and poly(A) RNAs. Nat Struct Mol Biol 11, 1206–1214 (2004). https://doi.org/10.1038/nsmb858

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