Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Structural analysis of kasugamycin inhibition of translation

Nature Structural & Molecular Biology volume 13, pages 879–886 (2006)Cite this article

Abstract

The prokaryotic ribosome is an important target of antibiotic action. We determined the X-ray structure of the aminoglycoside kasugamycin (Ksg) in complex with the Escherichia coli 70S ribosome at 3.5-Å resolution. The structure reveals that the drug binds within the messenger RNA channel of the 30S subunit between the universally conserved G926 and A794 nucleotides in 16S ribosomal RNA, which are sites of Ksg resistance. To our surprise, Ksg resistance mutations do not inhibit binding of the drug to the ribosome. The present structural and biochemical results indicate that inhibition by Ksg and Ksg resistance are closely linked to the structure of the mRNA at the junction of the peptidyl-tRNA and exit-tRNA sites (P and E sites).

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Location of the Ksg-binding pocket in the ribosome.
Figure 2: Determinants of resistance to Ksg in the DASL.
Figure 3: Chemical modification of mutant and wild-type 30S subunits in the presence of Ksg.
Figure 4: Toeprint experiments with leaderless mRNA.
Figure 5: Effect of Ksg on β-gal synthesis.
Figure 6: Dependence of inhibitory effect of Ksg on the primary sequence of the mRNA between positions −3 and +1.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

Referenced accessions

Protein Data Bank

References

  1. Laursen, B.S., Sorensen, H.P., Mortensen, K.K. & Sperling-Petersen, H.U. Initiation of protein synthesis in bacteria. Microbiol. Mol. Biol. Rev. 69, 101–123 (2005).

    Article  CAS  Google Scholar 

  2. Kozak, M. Regulation of translation via mRNA structure in prokaryotes and eukaryotes. Gene 361, 13–37 (2005).

    Article  CAS  Google Scholar 

  3. Boni, I.V., Isaeva, D.M., Musychenko, M.L. & Tzareva, N.V. Ribosome-messenger recognition: mRNA target sites for ribosomal protein S1. Nucleic Acids Res. 19, 155–162 (1991).

    Article  CAS  Google Scholar 

  4. Hui, A., Hayflick, J., Dinkelspiel, K. & de Boer, H.A. Mutagenesis of the three bases preceding the start codon of the beta-galactosidase mRNA and its effect on translation in Escherichia coli. EMBO J. 3, 623–629 (1984).

    Article  CAS  Google Scholar 

  5. Matteucci, M.D. & Heyneker, H.L. Targeted random mutagenesis: the use of ambiguously synthesized oligonucleotides to mutagenize sequences immediately 5′ of an ATG initiation codon. Nucleic Acids Res. 11, 3113–3121 (1983).

    Article  CAS  Google Scholar 

  6. Carter, A.P. et al. Functional insights from the structure of the 30S ribosomal subunit and its interactions with antibiotics. Nature 407, 340–348 (2000).

    Article  CAS  Google Scholar 

  7. Yusupova, G.Z., Yusupov, M.M., Cate, J.H. & Noller, H.F. The path of messenger RNA through the ribosome. Cell 106, 233–241 (2001).

    Article  CAS  Google Scholar 

  8. Okuyama, A., Machiyama, N., Kinoshita, T. & Tanaka, N. Inhibition by kasugamycin of initiation complex formation on 30S ribosomes. Biochem. Biophys. Res. Commun. 43, 196–199 (1971).

    Article  CAS  Google Scholar 

  9. Okuyama, A. & Tanaka, N. Differential effects of aminoglycosides on cistron-specific initiation of protein synthesis. Biochem. Biophys. Res. Commun. 49, 951–957 (1972).

    Article  CAS  Google Scholar 

  10. Poldermans, B., Goosen, N. & Van Knippenberg, P.H. Studies on the function of two adjacent N6,N6-dimethyladenosines near the 3′ end of 16 S ribosomal RNA of Escherichia coli. I. The effect of kasugamycin on initiation of protein synthesis. J. Biol. Chem. 254, 9085–9089 (1979).

    CAS  PubMed  Google Scholar 

  11. Tai, P.C., Wallace, B.J. & Davis, B.D. Actions of aurintricarboxylate, kasugamycin, and pactamycin on Escherichia coli polysomes. Biochemistry 12, 616–620 (1973).

    Article  CAS  Google Scholar 

  12. Kozak, M. & Nathans, D. Differential inhibition of coliphage MS2 protein synthesis by ribosome-directed antibiotics. J. Mol. Biol. 70, 41–55 (1972).

    Article  CAS  Google Scholar 

  13. Umezawa, H., Hamada, M., Suhara, Y., Hashimoto, T. & Ikekawa, T. Kasugamycin, a new antibiotic. Antimicrob. Agents Chemother. 5, 753–757 (1965).

    CAS  PubMed  Google Scholar 

  14. Ishigami, J., Fukuda, Y. & Hara, S. Clinical use of Kasugamycin for urinary tract infections due to Pseudomonas aeruginosa. J. Antibiot. [B] 20, 83–84 (1967).

    CAS  Google Scholar 

  15. Sparling, P.F. Kasugamycin resistance: 30S ribosomal mutation with an unusual location on the Escherichia coli chromosome. Science 167, 56–58 (1970).

    Article  CAS  Google Scholar 

  16. Helser, T.L., Davies, J.E. & Dahlberg, J.E. Mechanism of kasugamycin resistance in Escherichia coli. Nat. New Biol. 235, 6–9 (1972).

    Article  CAS  Google Scholar 

  17. Baan, R.A. et al. High-resolution proton magnetic resonance study of the secondary structure of the 3′-terminal 49-nucleotide fragment of 16S rRNA from Escherichia coli. Proc. Natl. Acad. Sci. USA 74, 1028–1031 (1977).

    Article  CAS  Google Scholar 

  18. O'Farrell, H.C., Scarsdale, J.N. & Rife, J.P. Crystal structure of KsgA, a universally conserved rRNA adenine dimethyltransferase in Escherichia coli. J. Mol. Biol. 339, 337–353 (2004).

    Article  CAS  Google Scholar 

  19. Vila-Sanjurjo, A., Squires, C.L. & Dahlberg, A.E. Isolation of kasugamycin resistant mutants in the 16 S ribosomal RNA of Escherichia coli. J. Mol. Biol. 293, 1–8 (1999).

    Article  CAS  Google Scholar 

  20. Woodcock, J., Moazed, D., Cannon, M., Davies, J. & Noller, H.F. Interaction of antibiotics with A- and P-site-specific bases in 16S ribosomal RNA. EMBO J. 10, 3099–3103 (1991).

    Article  CAS  Google Scholar 

  21. Schuwirth, B.S. et al. Structures of the bacterial ribosome at 3.5 Å resolution. Science 310, 827–834 (2005).

    Article  CAS  Google Scholar 

  22. Moazed, D. & Noller, H.F. Binding of tRNA to the ribosomal A and P sites protects two distinct sets of nucleotides in 16 S rRNA. J. Mol. Biol. 211, 135–145 (1990).

    Article  CAS  Google Scholar 

  23. Yusupov, M.M. et al. Crystal structure of the ribosome at 5.5 Å resolution. Science 292, 883–896 (2001).

    Article  CAS  Google Scholar 

  24. Cunningham, P.R. et al. Site-specific mutation of the conserved m26A m26A residues of E. coli 16S ribosomal RNA. Effects on ribosome function and activity of the ksgA methyltransferase. Biochim. Biophys. Acta 1050, 18–26 (1990).

    Article  CAS  Google Scholar 

  25. Vila-Sanjurjo, A. & Dahlberg, A.E. Mutational analysis of the conserved bases C1402 and A1500 in the center of the decoding domain of Escherichia coli 16 S rRNA reveals an important tertiary interaction. J. Mol. Biol. 308, 457–463 (2001).

    Article  CAS  Google Scholar 

  26. Moll, I. & Blasi, U. Differential inhibition of 30S and 70S translation initiation complexes on leaderless mRNA by kasugamycin. Biochem. Biophys. Res. Commun. 297, 1021–1026 (2002).

    Article  CAS  Google Scholar 

  27. Chin, K., Shean, C.S. & Gottesman, M.E. Resistance of lambda cI translation to antibiotics that inhibit translation initiation. J. Bacteriol. 175, 7471–7473 (1993).

    Article  CAS  Google Scholar 

  28. Andre, A. et al. Reinitiation of protein synthesis in Escherichia coli can be induced by mRNA cis-elements unrelated to canonical translation initiation signals. FEBS Lett. 468, 73–78 (2000).

    Article  CAS  Google Scholar 

  29. O'Donnell, S.M. & Janssen, G.R. The initiation codon affects ribosome binding and translational efficiency in Escherichia coli of cI mRNA with or without the 5′ untranslated leader. J. Bacteriol. 183, 1277–1283 (2001).

    Article  CAS  Google Scholar 

  30. Okuyama, A., Tanaka, N. & Komai, T. The binding of kasugamycin to the Escherichia coli ribosomes. J. Antibiot. (Tokyo) 28, 903–905 (1975).

    Article  CAS  Google Scholar 

  31. Cannone, J.J. et al. The comparative RNA web (CRW) site: an online database of comparative sequence and structure information for ribosomal, intron, and other RNAs. BMC Bioinformatics 3, 2 (2002).

    Article  Google Scholar 

  32. Allen, P.N. & Noller, H.F. Mutations in ribosomal proteins S4 and S12 influence the higher order structure of 16 S ribosomal RNA. J. Mol. Biol. 208, 457–468 (1989).

    Article  CAS  Google Scholar 

  33. Lowary, P., Sampson, J., Milligan, J., Groebe, D. & Uhlenbeck, O.C. in Structure and dynamics of RNA (eds Knippenberg, P.H. & Hilbers, C.W.) 69–76 (Plenum, New York, 1986).

    Book  Google Scholar 

  34. Marquez, V., Wilson, D.N., Tate, W.P., Triana-Alonso, F. & Nierhaus, K.H. Maintaining the ribosomal reading frame: the influence of the E site during translational regulation of release factor 2. Cell 118, 45–55 (2004).

    Article  CAS  Google Scholar 

  35. Shine, J. & Dalgarno, L. The 3′-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. Proc. Natl. Acad. Sci. USA 71, 1342–1346 (1974).

    Article  CAS  Google Scholar 

  36. Calogero, R.A., Pon, C.L., Canonaco, M.A. & Gualerzi, C.O. Selection of the mRNA translation initiation region by Escherichia coli ribosomes. Proc. Natl. Acad. Sci. USA 85, 6427–6431 (1988).

    Article  CAS  Google Scholar 

  37. Studer, S.M. & Joseph, S. Unfolding of mRNA secondary structure by the bacterial translation initiation complex. Mol. Cell 22, 105–115 (2006).

    Article  CAS  Google Scholar 

  38. Kozak, M. At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells. J. Mol. Biol. 196, 947–950 (1987).

    Article  CAS  Google Scholar 

  39. Allen, G.S., Zavialov, A., Gursky, R., Ehrenberg, M. & Frank, J. The cryo-EM structure of a translation initiation complex from Escherichia coli. Cell 121, 703–712 (2005).

    Article  CAS  Google Scholar 

  40. Brunger, A.T. et al. Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. D Biol. Crystallogr. 54, 905–921 (1998).

    Article  CAS  Google Scholar 

  41. Cowtan, K. General quadratic functions in real and reciprocal space and their application to likelihood phasing. Acta Crystallogr. D Biol. Crystallogr. 56, 1612–1621 (2000).

    Article  CAS  Google Scholar 

  42. Ikekawa, T., Umezawa, H. & Iitaka, Y. The structure of kasugamycin hydrobromide by x-ray crystallographic analysis. J. Antibiot. (Tokyo) 19, 49–50 (1966).

    CAS  Google Scholar 

  43. Thompson, M.A. ArgusLab 4.0.1 (Planaria Software, Seattle, 2004).

  44. Jones, T.A., Zou, J.Y., Cowan, S.W. & Kjeldgaard, M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119 (1991).

    Article  Google Scholar 

  45. Vila, A., Viril-Farley, J. & Tapprich, W.E. Pseudoknot in the central domain of small subunit ribosomal RNA is essential for translation. Proc. Natl. Acad. Sci. USA 91, 11148–11152 (1994).

    Article  CAS  Google Scholar 

  46. Vila-Sanjurjo, A. et al. X-ray crystal structures of the WT and a hyper-accurate ribosome from Escherichia coli. Proc. Natl. Acad. Sci. USA 100, 8682–8687 (2003).

    Article  CAS  Google Scholar 

  47. Van Etten, W.J. & Janssen, G.R. An AUG initiation codon, not codon-anticodon complementarity, is required for the translation of unleadered mRNA in Escherichia coli. Mol. Microbiol. 27, 987–1001 (1998).

    Article  CAS  Google Scholar 

  48. Miller, J.H. A Short Course in Bacterial Genetics (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA, 1991).

    Google Scholar 

Download references

Acknowledgements

We thank K. Frankel, G. Hura and J. Holton for help with data measurements at the SIBYLS beamline at the Advanced Light Source. A.V.-S. would like to acknowledge I. Tinoco for valuable discussions, C.M. Brown for help with Transterm and F. Vila for balance and coherence. This work was funded by the US National Institutes of Health (GM65050 to J.H.D.C., GM065120 to G.R.J, GM19756 to A.E.D. and National Cancer Institute grant CA92584 for the SIBYLS and 8.3.1 beamlines) and by the US Department of Energy (DE-AC03-76SF00098, KP110201 and LBNL LDRD 366851 to J.H.D.C. and DE-AC03 76SF00098 for the SIBYLS and 8.3.1 beamlines).

Author information

Author notes

  1. Barbara S Schuwirth

    Present address: Cancer research UK London Research Institute, Clare Hall Laboratories, South Mimms, Hertfordshire, EN6 3LD, UK

  2. J Michael Day

    Present address: US Department of Agriculture, Agricultural Research Service, SEPRL, 934 College Station Road, Athens, Georgia, 30605, USA

  3. Cathy W Hau

    Present address: Department of Anesthesia, University of California, San Francisco, California, 94143, USA

  4. Antón Vila-Sanjurjo

    Present address: Berkeley Center for Synthetic Biology, University of California, 717 Potter St., Berkeley, California, 94720-3224, USA

Authors and Affiliations

  1. Departments of Molecular and Cell Biology and Chemistry, University of California, Berkeley, 94720, California, USA

    Barbara S Schuwirth, Cathy W Hau, Jamie H Doudna Cate & Antón Vila-Sanjurjo

  2. Department of Microbiology, Miami University, Oxford, 45056, Ohio, USA

    J Michael Day & Gary R Janssen

  3. Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, 02912, Rhode Island, USA

    Albert E Dahlberg

  4. Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, 94720, California, USA

    Jamie H Doudna Cate & Antón Vila-Sanjurjo

Authors
  1. Barbara S Schuwirth
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. J Michael Day
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Cathy W Hau
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Gary R Janssen
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Albert E Dahlberg
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jamie H Doudna Cate
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Antón Vila-Sanjurjo
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Jamie H Doudna Cate or Antón Vila-Sanjurjo.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Binding of Ksg to the ribosome. (PDF 702 kb)

Supplementary Fig. 2

Total relative activities of NUG mRNA constructs. (PDF 724 kb)

Supplementary Table 1

Data collection and refinement statistics. (PDF 71 kb)

Supplementary Table 2

Quantification of Ksg footprints. (PDF 61 kb)

Supplementary Table 3

Quantification of toeprints. (PDF 68 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Schuwirth, B., Day, J., Hau, C. et al. Structural analysis of kasugamycin inhibition of translation. Nat Struct Mol Biol 13, 879–886 (2006). https://doi.org/10.1038/nsmb1150

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb1150

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing