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Functional insights from the structure of the 30S ribosomal subunit and its interactions with antibiotics

Abstract

The 30S ribosomal subunit has two primary functions in protein synthesis. It discriminates against aminoacyl transfer RNAs that do not match the codon of messenger RNA, thereby ensuring accuracy in translation of the genetic message in a process called decoding. Also, it works with the 50S subunit to move the tRNAs and associated mRNA by precisely one codon, in a process called translocation. Here we describe the functional implications of the high-resolution 30S crystal structure presented in the accompanying paper, and infer details of the interactions between the 30S subunit and its tRNA and mRNA ligands. We also describe the crystal structure of the 30S subunit complexed with the antibiotics paromomycin, streptomycin and spectinomycin, which interfere with decoding and translocation. This work reveals the structural basis for the action of these antibiotics, and leads to a model for the role of the universally conserved 16S RNA residues A1492 and A1493 in the decoding process.

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Figure 1: Overview of the 30S A-, P- and E-sites.
Figure 2: Detailed views of the 30S A-, P-, and E-sites with models of bound tRNA and mRNA ligands.
Figure 3: The H27 switch and its environment in the 30S subunit.
Figure 4: Interaction of spectinomycin with the 30S ribosomal subunit.
Figure 5: Interaction of streptomycin with the 30S ribosomal subunit.
Figure 6: Interaction of paromomycin with the 30S ribosomal subunit.
Figure 7: Model for the role of A1492 and A1493 in decoding.

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References

  1. Green, R. & Noller, H. F. Ribosomes and translation. Annu. Rev. Biochem. 66, 679–716 (1997).

    Article  CAS  Google Scholar 

  2. Agrawal, R. K. et al. Direct visualization of A-, P-, and E-site transfer RNAs in the Escherichia coli ribosome. Science 271, 1000–1002 (1996).

    Article  ADS  CAS  Google Scholar 

  3. Stark, H. et al. Arrangement of tRNAs in pre- and posttranslocational ribosomes revealed by electron cryomicroscopy. Cell 88, 19–28 (1997).

    Article  CAS  Google Scholar 

  4. Kurland, C. G. Translational accuracy and the fitness of bacteria. Annu. Rev. Genet. 26, 29–50 ( 1992).

    Article  CAS  Google Scholar 

  5. Pape, T., Wintermeyer, W. & Rodnina, M. V. Complete kinetic mechanism of elongation factor Tu-dependent binding of aminoacyl-tRNA to the A site of the E. coli ribosome. EMBO J. 17, 7490–7497 ( 1998).

    Article  CAS  Google Scholar 

  6. Pape, T., Wintermeyer, W. & Rodnina, M. Induced fit in initial selection and proof-reading of aminoacyl-tRNA on the ribosome. EMBO J. 18, 3800–3807 (1999).

    Article  CAS  Google Scholar 

  7. Pape, T., Wintermeyer, W. & Rodnina, M. V. Conformational switch in the decoding region of 16S rRNA during aminoacyl-tRNA selection on the ribosome. Nature Struct. Biol. 7, 104–107 ( 2000).

    Article  CAS  Google Scholar 

  8. Gabashvili, I. S. et al. Major rearrangements in the 70S ribosomal 3D structure caused by a conformational switch in 16S ribosomal RNA. EMBO J. 18, 6501–6507 (1999).

    Article  CAS  Google Scholar 

  9. Nierhaus, K. H. The allosteric three-site model for the ribosomal elongation cycle: features and future. Biochemistry 29, 4997– 5008 (1990).

    Article  CAS  Google Scholar 

  10. 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 

  11. Lodmell, J. S. & Dahlberg, A. E. A conformational switch in Escherichia coli 16S ribosomal RNA during decoding of messenger RNA. Science 277, 1262– 1267 (1997).

    Article  CAS  Google Scholar 

  12. Wimberly, B. T. et al. Structure of the 30S ribosomal subunit. Nature 407, 327–339 ( 2000).

    Article  ADS  CAS  Google Scholar 

  13. Mueller, F. & Brimacombe, R. A new model for the three-dimensional folding of Escherichia coli 16 S ribosomal RNA. I. Fitting the RNA to a 3D electron microscopic map at 20 Å. J. Mol. Biol. 271, 524–544 (1997).

    Article  CAS  Google Scholar 

  14. Cate, J. H., Yusupov, M. M., Yusupova, G. Z., Earnest, T. N. & Noller, H. F. X-ray crystal structures of 70S ribosome functional complexes. Science 285, 2095–2104 (1999).

    Article  CAS  Google Scholar 

  15. Frank, J. & Agrawal, R. K. A ratchet-like inter-subunit reorganization of the ribosome during translocation. Nature 406, 319–322 (2000).

    Article  ADS  Google Scholar 

  16. Stark, H., Rodnina, M. V., Wieden, H. J., van Heel, M. & Wintermeyer, W. Large-scale movement of elongation factor G and extensive conformational change of the ribosome during translocation. Cell 100, 301–309 (2000).

    Article  CAS  Google Scholar 

  17. 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 

  18. Prince, J. B., Taylor, B. H., Thurlow, D. L., Ofengand, J. & Zimmermann, R. A. Covalent crosslinking of tRNA1Val to 16S RNA at the ribosomal P site: identification of crosslinked residues. Proc. Natl Acad. Sci. USA 79, 5450– 5454 (1982).

    Article  ADS  CAS  Google Scholar 

  19. von Ahsen, U. & Noller, H. F. Identification of bases in 16S rRNA essential for tRNA binding at the 30S ribosomal P site. Science 267, 234–237 ( 1995).

    Article  ADS  CAS  Google Scholar 

  20. Mueller, F., Stark, H., van Heel, M., Rinke-Appel, J. & Brimacombe, R. A new model for the three-dimensional folding of Escherichia coli 16 S ribosomal RNA. III. The topography of the functional centre. J. Mol. Biol. 271, 566– 587 (1997).

    Article  CAS  Google Scholar 

  21. Garrett, R. A. (eds) The Ribosome. Structure, Function, Antibiotics and Cellular Interactions (ASM, Washington DC, 2000).

    Google Scholar 

  22. Kurland, C. G., Hughes, D., Ehrenberg, M. in Limitations of Translational Accuracy (eds Neidhardt, F. C. et al.) 979–1003 (ASM, Washington DC, 1996).

    Google Scholar 

  23. Bilgin, N., Richter, A. A., Ehrenberg, M., Dahlberg, A. E. & Kurland, C. G. Ribosomal RNA and protein mutants resistant to spectinomycin. EMBO J. 9, 735 –739 (1990).

    Article  CAS  Google Scholar 

  24. Moazed, D. & Noller, H. F. Interaction of antibiotics with functional sites in 16S ribosomal RNA. Nature 327, 389–394 (1987).

    Article  ADS  CAS  Google Scholar 

  25. Brink, M. F., Brink, G., Verbeet, M. P. & de Boer, H. A. Spectinomycin interacts specifically with the residues G1064 and C1192 in 16S rRNA, thereby potentially freezing this molecule into an inactive conformation. Nucleic Acids Res. 22, 325– 331 (1994).

    Article  CAS  Google Scholar 

  26. Wittmann-Liebold, B. & Greuer, B. The primary structure of protein S5 from the small subunit of the Escherichia coli ribosome. FEBS Lett. 95, 91–98 (1978).

    Article  CAS  Google Scholar 

  27. Ruusala, T. & Kurland, C. G. Streptomycin preferentially perturbs ribosomal proofreading. Mol. Gen. Genet. 198, 100–104 (1984).

    Article  CAS  Google Scholar 

  28. Karimi, R. & Ehrenberg, M. Dissociation rate of cognate peptidyl-tRNA from the A-site of hyper- accurate and error-prone ribosomes. Eur. J. Biochem. 226, 355–360 (1994).

    Article  CAS  Google Scholar 

  29. Karimi, R. & Ehrenberg, M. Dissociation rates of peptidyl-tRNA from the P-site of E. coli ribosomes. EMBO J. 15, 1149–1154 (1996).

    Article  CAS  Google Scholar 

  30. Bilgin, N. & Ehrenberg, M. Mutations in 23 S ribosomal RNA perturb transfer RNA selection and can lead to streptomycin dependence. J. Mol. Biol. 235, 813–824 (1994).

    Article  CAS  Google Scholar 

  31. Gravel, M., Melancon, P. & Brakier-Gingras, L. Cross-linking of streptomycin to the 16S ribosomal RNA of Escherichia coli. Biochemistry 26, 6227–6232 (1987).

    Article  CAS  Google Scholar 

  32. Montandon, P. E., Wagner, R. & Stutz, E. E. coli ribosomes with a C912 to U base change in the 16S rRNA are streptomycin resistant. EMBO J. 5, 3705–3708 (1986).

    Article  CAS  Google Scholar 

  33. Pinard, R., Payant, C., Melancon, P. & Brakier-Gingras, L. The 5′ proximal helix of 16S rRNA is involved in the binding of streptomycin to the ribosome. FASEB J. 7, 173– 176 (1993).

    Article  CAS  Google Scholar 

  34. Melancon, P., Lemieux, C. & Brakier-Gingras, L. A mutation in the 530 loop of Escherichia coli 16S ribosomal RNA causes resistance to streptomycin. Nucleic Acids Res. 16, 9631–9639 ( 1988).

    Article  CAS  Google Scholar 

  35. Montandon, P. E., Nicolas, P., Schurmann, P. & Stutz, E. Streptomycin–resistance of Euglena gracilis chloroplasts: identification of a point mutation in the 16S rRNA gene in an invariant position. Nucleic Acids Res. 13, 4299–4310 (1985).

    Article  CAS  Google Scholar 

  36. Leclerc, D., Melancon, P. & Brakier-Gingras, L. Mutations in the 915 region of Escherichia coli 16S ribosomal RNA reduce the binding of streptomycin to the ribosome. Nucleic Acids Res. 19, 3973–3977 (1991).

    Article  CAS  Google Scholar 

  37. Melancon, P., Boileau, G. & Brakier-Gingras, L. Cross-linking of streptomycin to the 30S subunit of Escherichia coli with phenyldiglyoxal. Biochemistry 23, 6697–6703 (1984).

    Article  CAS  Google Scholar 

  38. Powers, T. & Noller, H. F. A functional pseudoknot in 16S ribosomal RNA. EMBO J. 10, 2203– 2214 (1991).

    Article  CAS  Google Scholar 

  39. Bock, A., Petzet, A. & Piepersberg, W. Ribosomal ambiguity (ram) mutations facilitate diyhydrostreptomycin binding to ribosomes. FEBS Lett. 104, 317 –321 (1979).

    Article  CAS  Google Scholar 

  40. Spahn, C. M. & Prescott, C. D. Throwing a spanner in the works: antibiotics and the translation apparatus. J. Mol. Med. 74, 423–439 (1996).

    Article  CAS  Google Scholar 

  41. Fourmy, D., Recht, M. I., Blanchard, S. C. & Puglisi, J. D. Structure of the A site of Escherichia coli 16S ribosomal RNA complexed with an aminoglycoside antibiotic. Science 274, 1367–1371 (1996).

    Article  ADS  CAS  Google Scholar 

  42. Yoshizawa, S., Fourmy, D. & Puglisi, J. D. Recognition of the codon–anticodon helix by ribosomal RNA. Science 285, 1722– 1725 (1999).

    Article  CAS  Google Scholar 

  43. VanLoock, M. S., Easterwood, T. R. & Harvey, S. C. Major groove binding of the tRNA/mRNA complex to the 16 S ribosomal RNA decoding site. J. Mol. Biol. 285, 2069–2078 (1999).

    Article  CAS  Google Scholar 

  44. Otwinowski, Z. & Minor, W. in Methods in Enzymology (eds Carter, C. W. J. & Sweet, R. M.) 307– 325 (Academic, New York, 1997).

    Google Scholar 

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

    Article  Google Scholar 

  46. Carson, M. Ribbons 2. 0. J. Appl. Cryst. 24, 958– 961 (1991).

    Article  Google Scholar 

Download references

Acknowledgements

This work was supported by the Medical Research Council (UK) and a grant from the US National Institutes of Health to V.R. and S.W.White. D.E.B. was supported by an EMBO long-term fellowship, and W.M.C. by an NIH predoctoral fellowship. We thank R. Ravelli for help with data collection, and M. Pacold for help and discussions with the modelling of antibiotic structures.

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Correspondence to V. Ramakrishnan.

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Carter, A., Clemons, W., Brodersen, D. et al. Functional insights from the structure of the 30S ribosomal subunit and its interactions with antibiotics. Nature 407, 340–348 (2000). https://doi.org/10.1038/35030019

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