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:

An assembly landscape for the 30S ribosomal subunit

Nature volume 438, pages 628–632 (2005)Cite this article

Abstract

Self-assembling macromolecular machines drive fundamental cellular processes, including transcription, messenger RNA processing, translation, DNA replication and cellular transport. The ribosome, which carries out protein synthesis, is one such machine, and the 30S subunit of the bacterial ribosome is the preeminent model system for biophysical analysis of large RNA–protein complexes. Our understanding of 30S assembly is incomplete, owing to the challenges of monitoring the association of many components simultaneously. Here we have developed a method involving pulse–chase monitored by quantitative mass spectrometry (PC/QMS) to follow the assembly of the 20 ribosomal proteins with 16S ribosomal RNA during formation of the functional particle. These data represent a detailed and quantitative kinetic characterization of the assembly of a large multicomponent macromolecular complex. By measuring the protein binding rates at a range of temperatures, we find that local transformations throughout the assembling subunit have similar but distinct activation energies. Thus, the prevailing view of 30S assembly as a pathway proceeding through a global rate-limiting conformational change must give way to one in which the assembly of the complex traverses a landscape dotted with various local conformational transitions.

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: The PC/QMS method for measuring protein binding kinetics in the 30S ribosomal subunit.
Figure 2: Binding kinetics of 30S proteins measured using PC/QMS under standard conditions.
Figure 3: Ratio of the protein binding rates observed at two concentrations versus the rates at standard concentration.
Figure 4: The temperature dependence of protein binding rates.
Figure 5: An assembly landscape for 30S assembly.

Similar content being viewed by others

References

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

    Article  ADS  CAS  Google Scholar 

  2. Schluenzen, F. et al. Structure of functionally activated small ribosomal subunit at 3.3 Å resolution. Cell 102, 615–623 (2000)

    Article  CAS  Google Scholar 

  3. Brodersen, D. E., Clemons, W. M. Jr, Carter, A. P., Wimberly, B. T. & Ramakrishnan, V. Crystal structure of the 30 S ribosomal subunit from Thermus thermophilus: structure of the proteins and their interactions with 16 S RNA. J. Mol. Biol. 316, 725–768 (2002)

    Article  CAS  Google Scholar 

  4. Moazed, D., Stern, S. & Noller, H. F. Rapid chemical probing of conformation in 16 S ribosomal RNA and 30 S ribosomal subunits using primer extension. J. Mol. Biol. 187, 399–416 (1986)

    Article  CAS  Google Scholar 

  5. Agalarov, S. C. & Williamson, J. R. A hierarchy of RNA subdomains in assembly of the central domain of the 30 S ribosomal subunit. RNA 6, 402–408 (2000)

    Article  CAS  Google Scholar 

  6. Powers, T., Daubresse, G. & Noller, H. F. Dynamics of in vitro assembly of 16 S rRNA into 30 S ribosomal subunits. J. Mol. Biol. 232, 362–374 (1993)

    Article  CAS  Google Scholar 

  7. Recht, M. I. & Williamson, J. R. Thermodynamics and kinetics of central domain assembly. Cold Spring Harbor Symp. Quant. Biol. 66, 591–598 (2001)

    Article  CAS  Google Scholar 

  8. Stagg, S. M., Mears, J. A. & Harvey, S. C. A structural model for the assembly of the 30S subunit of the ribosome. J. Mol. Biol. 328, 49–61 (2003)

    Article  CAS  Google Scholar 

  9. Stern, S., Powers, T., Changchien, L. M. & Noller, H. F. RNA–protein interactions in 30S ribosomal subunits: folding and function of 16S rRNA. Science 244, 783–790 (1989)

    Article  ADS  CAS  Google Scholar 

  10. Held, W. A., Ballou, B., Mizushima, S. & Nomura, M. Assembly mapping of 30 S ribosomal proteins from Escherichia coli. J. Biol. Chem. 249, 3103–3111 (1974)

    CAS  PubMed  Google Scholar 

  11. Culver, G. M. Assembly of the 30S ribosomal subunit. Biopolymers 68, 234–249 (2003)

    Article  CAS  Google Scholar 

  12. Traub, P. & Nomura, M. Structure and function of Escherichia coli ribosomes VI. Mechanism of assembly of 30 S ribosomes studied in vitro. J. Mol. Biol. 40, 391–413 (1969)

    Article  CAS  Google Scholar 

  13. Held, W. A. & Nomura, M. Rate-determining step in the reconstitution of Escherichia coli 30S ribosomal subunits. Biochemistry 12, 3273–3281 (1973)

    Article  CAS  Google Scholar 

  14. Holmes, K. L. & Culver, G. M. Mapping structural differences between 30S ribosomal subunit assembly intermediates. Nature Struct. Mol. Biol. 11, 179–186 (2004)

    Article  CAS  Google Scholar 

  15. Gygi, S. P. et al. Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nature Biotechnol. 17, 994–999 (1999)

    Article  CAS  Google Scholar 

  16. Oda, Y., Huang, K., Cross, F. R., Cowburn, D. & Chait, B. T. Accurate quantitation of protein expression and site-specific phosphorylation. Proc. Natl Acad. Sci. USA 96, 6591–6596 (1999)

    Article  ADS  CAS  Google Scholar 

  17. Pasa-Tolic, L. et al. High throughput proteome-wide precision measurements of protein expression using mass spectrometry. J. Am. Chem. Soc. 121, 7949–7950 (1999)

    Article  CAS  Google Scholar 

  18. Sechi, S. & Oda, Y. Quantitative proteomics using mass spectrometry. Curr. Opin. Chem. Biol. 7, 70–77 (2003)

    Article  CAS  Google Scholar 

  19. Traub, P. & Nomura, M. Structure and function of E. coli ribosomes. V. Reconstitution of functionally active 30S ribosomal particles from RNA and proteins. Proc. Natl Acad. Sci. USA 59, 777–784 (1968)

    Article  ADS  CAS  Google Scholar 

  20. Beavis, R. C. & Chait, B. T. Matrix-assisted laser desorption ionization mass-spectrometry of proteins. Methods Enzymol. 270, 519–551 (1996)

    Article  CAS  Google Scholar 

  21. Hillenkamp, F. & Karas, M. Mass spectrometry of peptides and proteins by matrix-assisted ultraviolet laser desorption/ionization. Methods Enzymol. 193, 280–295 (1990)

    Article  CAS  Google Scholar 

  22. Jennings, K. R. & Dolnikowski, G. G. Mass analyzers. Methods Enzymol. 193, 37–61 (1990)

    Article  CAS  Google Scholar 

  23. Pichon, J., Marvaldi, J. & Marchis-Mouren, G. The in vivo order of protein addition in the course of Escherichia coli 30 S and 50 S subunit biogenesis. J. Mol. Biol. 96, 125–137 (1975)

    Article  CAS  Google Scholar 

  24. Mangiarotti, G., Apirion, D., Schlessinger, D. & Silengo, L. Biosynthetic precursors of 30S and 50S ribosomal particles in Escherichia coli. Biochemistry 7, 456–472 (1968)

    Article  CAS  Google Scholar 

  25. Srivastava, A. K. & Schlessinger, D. in The Ribosome: Structure, Function, & Evolution (ed. Hill, W. et al.) 426–434 (American Society of Microbiology, Washington DC, 1990)

    Google Scholar 

  26. Xia, T. et al. Thermodynamic parameters for an expanded nearest-neighbour model for formation of RNA duplexes with Watson–Crick base pairs. Biochemistry 37, 14719–14735 (1998)

    Article  CAS  Google Scholar 

  27. Dill, K. A. & Chan, H. S. From Levinthal to pathways to funnels. Nature Struct. Biol. 4, 10–19 (1997)

    Article  CAS  Google Scholar 

  28. Pan, J., Thirumalai, D. & Woodson, S. A. Folding of RNA involves parallel pathways. J. Mol. Biol. 273, 7–13 (1997)

    Article  CAS  Google Scholar 

  29. Rook, M. S., Treiber, D. K. & Williamson, J. R. Fast folding mutants of the Tetrahymena group I ribozyme reveal a rugged folding energy landscape. J. Mol. Biol. 281, 609–620 (1998)

    Article  CAS  Google Scholar 

  30. Thirumalai, D. & Woodson, S. A. Kinetics of folding of proteins and RNA. Acc. Chem. Res. 29, 433–439 (1996)

    Article  CAS  Google Scholar 

  31. Nomura, M. Assembly of bacterial ribosomes. Science 179, 864–873 (1973)

    Article  ADS  CAS  Google Scholar 

  32. Alix, J. H. & Guerin, M. F. Mutant DnaK chaperones cause ribosome assembly defects in Escherichia coli. Proc. Natl Acad. Sci. USA 90, 9725–9729 (1993)

    Article  ADS  CAS  Google Scholar 

  33. Maki, J. A., Schnobrich, D. J. & Culver, G. M. The DnaK chaperone system facilitates 30S ribosomal subunit assembly. Mol. Cell 10, 129–138 (2002)

    Article  CAS  Google Scholar 

  34. Maki, J. A., Southworth, D. R. & Culver, G. M. Demonstration of the role of the DnaK chaperone system in assembly of 30S ribosomal subunits using a purified in vitro system. RNA 9, 1418–1421 (2003)

    Article  CAS  Google Scholar 

  35. Serdyuk, I. N., Agalarov, S. C., Sedelnikova, S. E., Spirin, A. S. & May, R. P. Shape and compactness of the isolated ribosomal 16 S RNA and its complexes with ribosomal proteins. J. Mol. Biol. 169, 409–425 (1983)

    Article  CAS  Google Scholar 

  36. Culver, G. M. & Noller, H. F. Efficient reconstitution of functional Escherichia coli 30S ribosomal subunits from a complete set of recombinant small subunit ribosomal proteins. RNA 5, 832–843 (1999)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank the staff of the TSRI Center for Mass Spectrometry for assistance with mass spectrometry; M. I. Recht, S. C. Agalarov and S. P. Ryder for discussions and technical assistance; the laboratories of D. B. Goodin, S. P. Mayfield and A. Schneemann for use of equipment; and M. J. Fedor, J. D. Puglisi and S. P. Ryder for critically reading the manuscript. This work was supported by a grant from the NIH (to J.R.W.) and by predoctoral fellowships from the NSF and the Skaggs Institute for Chemical Biology (to M.W.T.T.). Author Contributions The experimental work in this manuscript was carried out by M.W.T.T., with advice and support from G.S. and J.R.W.

Author information

Author notes

  1. Megan W. T. Talkington

    Present address: Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts, 02115, USA

Authors and Affiliations

  1. Departments of Molecular Biology and Chemistry, and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, California, 92037, La Jolla, USA

    Megan W. T. Talkington, Gary Siuzdak & James R. Williamson

Authors
  1. Megan W. T. Talkington
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Gary Siuzdak
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. James R. Williamson
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to James R. Williamson.

Ethics declarations

Competing interests

Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The authors declare no competing financial interests.

Supplementary information

Supplementary Methods

Additional details of the methods used in this study. This file also contains additional references. (DOC 105 kb)

Supplementary Table S1

30S protein binding rates determined by PC/QMS at a range of temperatures and the resulting binding activation energies. (DOC 120 kb)

Supplementary Figure S1

Binding progress curves from PC/QMS and gel mobility shift for the S15:A4 model system. (PDF 2513 kb)

Supplementary Figure S2

30S protein binding progress curves from PC/QMS under standard conditions (as in Fig. 2a). Here the progress curves for the different proteins are shown (PDF 3321 kb)

Supplementary Figure S3

Arrhenius plots of the protein binding rates determined by PC/QMS at a range of temperatures (as in Fig. 4b). Here the Arrhenius plots for the different proteins are shown separately. (PDF 3878 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Talkington, M., Siuzdak, G. & Williamson, J. An assembly landscape for the 30S ribosomal subunit. Nature 438, 628–632 (2005). https://doi.org/10.1038/nature04261

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature04261

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

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