Article | Published:

Mapping structural differences between 30S ribosomal subunit assembly intermediates

Nature Structural & Molecular Biology volume 11, pages 179186 (2004) | Download Citation

Subjects

Abstract

Under appropriate conditions, functional Escherichia coli 30S ribosomal subunits assemble in vitro from purified components. However, at low temperatures, assembly stalls, producing an intermediate (RI) that sediments at 21S and is composed of 16S ribosomal RNA (rRNA) and a subset of ribosomal proteins (r-proteins). Incubation of RI at elevated temperatures produces a particle, RI*, of similar composition but different sedimentation coefficient (26S). Once formed, RI* rapidly associates with the remaining r-proteins to produce mature 30S subunits. To understand the nature of this transition from RI to RI*, changes in the reactivity of 16S rRNA between these two states were monitored by chemical modification and primer extension analysis. Evaluation of this data using structural and biochemical information reveals that many changes are r-protein–dependent and some are clustered in functional regions, suggesting that this transition is an important step in functional 30S subunit formation.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    & 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).

  2. 2.

    & Assembly mapping of 30S ribosomal proteins in E. coli. Nature 226, 1214–1218 (1970).

  3. 3.

    , & Reconstitution of Escherichia coli 30S ribosomal subunits from purified molecular components. J. Biol. Chem. 218, 5720–5730 (1973).

  4. 4.

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

  5. 5.

    , , & Assembly mapping of 30S ribosomal proteins from Escherichia coli. J. Biol. Chem. 249, 3103–3111 (1974).

  6. 6.

    & Structure and function of E. coli ribosomes. VI. Mechanism of assembly of 30S ribosomes studied in vitro. J. Mol. Biol. 40, 391–413 (1969).

  7. 7.

    & Rate determining step in the reconstitution of Escherichia coli 30S ribosomal subunits. Biochemistry 12, 3273–3281 (1973).

  8. 8.

    , & Structure and function of E. coli ribosomes. VIII. Cold-sensitive mutants defective in ribosome assembly. Proc. Natl. Acad. Sci. USA 63, 384–391 (1969).

  9. 9.

    , , & Structure and function of bacterial ribosomes. XII. Accumulation of 21S particles by some cold-sensitive mutants of E. coli. J. Mol. Biol. 62, 121–138 (1971).

  10. 10.

    , & Ribosomal proteins. XLIII. In vivo assembly of Escherichia coli ribosomal proteins. J. Mol. Biol. 74, 587–597 (1973).

  11. 11.

    Intermediates and time kinetics of the in vivo assembly of Escherichia coli ribosomes. J. Mol. Biol. 92, 15–37 (1975).

  12. 12.

    & Mutant DnaK chaperones cause ribosome assembly defects in Escherichia coli. Proc. Natl. Acad. Sci. USA 90, 9725–9729 (1993).

  13. 13.

    , , , & X-ray crystal structures of 70S ribosome functional complexes. Science 285, 2095–2104 (1999).

  14. 14.

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

  15. 15.

    , , & Interconversion of active and inactive 30 S ribosomal subunits is accompanied by a conformational change in the decoding region of 16S rRNA. J. Mol. Biol. 191, 483–493 (1986).

  16. 16.

    & Footprinting and modification-interference analysis of binding sites on RNA. In RNA:Protein Interactions. A Practical Approach (ed. Smith, C.W.J.) 237–253 (Oxford Univ. Press, New York, 1998).

  17. 17.

    & A functional pseudoknot in 16S ribosomal RNA. EMBO J. 10, 2203–2214 (1991).

  18. 18.

    , & Pseudoknot in the central domain of small subunit ribosomal RNA is essential for translation. Proc. Natl. Acad. Sci. USA 91, 11148–11152 (1994).

  19. 19.

    , , & Base complementarity in helix 2 of the central pseudoknot in 16S rRNA is essential for ribosome functioning. Nucleic Acids Res. 26, 549–553 (1998).

  20. 20.

    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).

  21. 21.

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

  22. 22.

    , & Construction and analysis of base-paired regions of the 16S rRNA in the 30S ribosomal subunit determined by constraint satisfaction molecular modeling. J. Mol. Graph. Model. 19, 495–513 (2001).

  23. 23.

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

  24. 24.

    et al. Crystal structure of the S15-rRNA complex. Nat. Struct. Biol. 7, 273–277 (2000).

  25. 25.

    , , , & Structure of the S15,S6,S18-rRNA complex: assembly of the 30S ribosome central domain. Science 288, 107–113 (2000).

  26. 26.

    , , , & Crystal structure of the 30 S ribosomal subunit from Thermus thermophilus: structure of the proteins and their interactions with 16S RNA. J. Mol. Biol. 316, 725–768 (2002).

  27. 27.

    & Hydroxyl radical footprinting of ribosomal proteins on 16S rRNA. RNA 1, 194–209 (1995).

  28. 28.

    , , & Interaction of proteins S16, S17 and S20 with 16S ribosomal RNA. J. Mol. Biol. 200, 291–299 (1988).

  29. 29.

    , , & Probing the assembly of the 3′ major domain of 16S ribosomal RNA. Quaternary interactions involving ribosomal proteins S7, S9 and S19. J. Mol. Biol. 200, 309–319 (1988).

  30. 30.

    , , & Probing the assembly of the 3′ major domain of 16S rRNA. Interactions involving ribosomal proteins S2, S3, S10, S13 and S14. J. Mol. Biol. 201, 697–716 (1988).

  31. 31.

    & Transfer RNA shields specific nucleotides in 16S ribosomal RNA from attack by chemical probes. Cell 47, 985–994 (1986).

  32. 32.

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

  33. 33.

    , , & The path of the messenger RNA through the ribosome. Cell 106, 233–241 (2001).

  34. 34.

    , , & Specific protection of 16S rRNA by translational initiation factors. J. Mol. Biol. 248, 207–210 (1995).

  35. 35.

    , & Rapid chemical probing of conformations in 16S ribosomal RNA and 30S ribosomal subunits using primer extension. J. Mol. Biol. 187, 399–416 (1986).

  36. 36.

    & In vitro reconstitution of 30S ribosomal subunits using complete set of recombinant proteins. Methods Enzymol. 318, 446–460 (2000).

  37. 37.

    Assembly of the 30S ribosomal subunit. Biopolymers 68, 234–249.

  38. 38.

    Ribbons. Methods Enzymol. 277, 493–505 (1997).

  39. 39.

    The structure of ribosomal RNA: A three-dimensional jigsaw puzzle. Eur. J. Biochem. 230, 365–383 (1995).

Download references

Acknowledgements

We thank R. Green, I. Jagannathan and J. Maki for critical reading of the manuscript. Additional thanks to S. Stagg and J. Hoy for assistance with figures. This work was funded by a grant from the US National Institutes of Health (to G.M.C.).

Author information

Affiliations

  1. Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, Iowa 50011, USA.

    • Kristi L Holmes
    •  & Gloria M Culver

Authors

  1. Search for Kristi L Holmes in:

  2. Search for Gloria M Culver in:

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Gloria M Culver.

Supplementary information

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nsmb719

Further reading