Skip to main content
SpringerLink
Log in

Characterization of a 30S ribosomal subunit assembly intermediate found in Escherichia coli cells growing with neomycin or paromomycin

  • Original Paper
  • Published:
Archives of Microbiology Aims and scope Submit manuscript

Abstract

Neomycin and paromomycin are aminoglycoside antibiotics that specifically stimulate the misreading of mRNA by binding to the decoding site of 16S rRNA in the 30S ribosomal subunit. Recent work has shown that both antibiotics also inhibit 30S subunit assembly in Escherichia coli and Staphylococcus aureus cells. This work describes the characteristics of an assembly intermediate produced in E. coli cells grown with neomycin or paromomycin. Antibiotic treatment stimulated the accumulation of a 30S assembly precursor with a sedimentation coefficient of 21S. The particle was able to bind radio-labeled antibiotics in vivo and in vitro. Hybridization experiments showed that the 21S precursor particle contained unprocessed 16S rRNA with both 5′ and 3′ extensions. Ten 30S ribosomal proteins were found in the precursor after inhibition by each drug. In addition, cell free reconstitution assays generated a 21S particle after incubation with either aminoglycoside. This work helps to define the features of the ribosome structure as a target for antimicrobial agents and may provide information needed for the design of more effective antibiotics.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  • Alix J-H (1993) Extrinsic factors in ribosome assembly. In: Nierhaus KH, Franceschi F, Subramanian AR, Erdmann VA, Wittmann-Liebold B (eds) The translational apparatus. Plenum, New York, pp 173–184

    Google Scholar 

  • Anderson RM (1999) The pandemic of antibiotic resistance. Nat Med 5:147–149

    Article  PubMed  CAS  Google Scholar 

  • Borovinskaya MA, Pai RD, Zhang W, Schuwirth BS, Holton JM, Hirokawa G, Kaji H, Kaji A, Cate JH (2007) Structural basis for aminoglycoside inhibition of bacterial ribosome recycling. Nat Struct Biol 14:727–732

    Article  CAS  Google Scholar 

  • Broderson DE, Clemons WM, Carter AP, Morgan-Warren RJ, Wimberly BT, Ramakrishnan V (2000) The structural basis for the action of the antibiotics tetracycline, pactamycin, and hygromycin B on the 30S ribosomal subunit. Cell 103:1143–1154

    Article  Google Scholar 

  • Brosius J, Dull TJ, Noller HF (1980) Complete nucleotide sequence of a 23S ribosomal RNA gene from Escherichia coli. Proc Natl Acad Sci US 77:201–204

    Article  CAS  Google Scholar 

  • Bryskier A (2005) Antimicrobial agents. Antibacterials and antifungals. American Society for Microbiology, Washington

    Google Scholar 

  • Carter AP, Clemons WM, Broderson DE, Morgan-Warren RJ, Wimberly BT, Ramakrishnan V (2000) Functional insights from the structure of the 30S ribosomal subunit and its interactions with antibiotics. Nature 407:340–348

    Article  PubMed  CAS  Google Scholar 

  • Champney WS (1989) Reductive methods for isotopic labeling of antibiotics. Anal Biochem 181:90–95

    Article  PubMed  CAS  Google Scholar 

  • Champney WS (2003) Bacterial ribosomal subunit assembly is an antibiotic target. Curr Top Med Chem 3:929–947

    Article  PubMed  CAS  Google Scholar 

  • Champney WS (2006) The other target for ribosomal antibiotics. Inhibition of bacterial ribosomal subunit formation. Infect Disord Drug Targets 6:377–390

    PubMed  CAS  Google Scholar 

  • Chu DT, Plattner JJ, Katz L (1996) New directions in antibacterial research. J Med Chem 39:3853–3874

    Article  PubMed  CAS  Google Scholar 

  • Culver GM (2003) Assembly of the 30S ribosomal subunit. Biopolymers 68:234–249

    Article  PubMed  CAS  Google Scholar 

  • Dahlberg AE, Horodyski F, Keller P (1978) Interaction of neomycin with ribosomes and ribosomal ribonucleic acid. Antimicrob Agents Chemother 13:331–339

    PubMed  CAS  Google Scholar 

  • Farrell RE (1993) RNA Methodologies. Academic, San Diego

    Google Scholar 

  • Fourmy D, Recht MI, Blanchard SC, Puglisi JD (1996) Structure of the A site of Escherichia coli 16S ribosomal RNA complexed with an aminoglycoside antibiotic. Science 274:1367–1371

    Article  PubMed  CAS  Google Scholar 

  • Fourmy D, Yoshizawa S, Puglisi JD (1998) Paromomycin binding induces a local conformational change in the A-site of 16 S rRNA. J Mol Biol 277:333–345

    Article  PubMed  CAS  Google Scholar 

  • Garrett RA, Douthwaite SR, Liljas A, Matheson AT, Moore PB, Noller HF (eds) (2000) The ribosome. Structure, function, antibiotics, and cellular interactions. ASM, Washington

  • Gesteland R (1966) Isolation and characterization of ribonuclease I mutants of Escherichia coli. J Mol Biol 16:67–84

    Article  PubMed  CAS  Google Scholar 

  • Geyl D, Bock A, Isono K (1981) An improved method for two-dimensional gel-electrophoresis: analysis of mutationally altered ribosomal proteins of Escherichia coli. Mol gen Genet 181:309–312

    Article  PubMed  CAS  Google Scholar 

  • Giri L, Hill WE, Wittmann HG (1984) Ribosomal proteins. In: Anfinsen CB, Edsall JT, Richards FM (eds) Advances in protein chemistry. Academic, New York, pp 1–78

    Google Scholar 

  • Hansen JL, Ippolito JA, Ban N, Nissen P, Moore PB, Steitz TA (2002) The structures of four macrolide antibiotics bound to the large ribosomal subunit. Mol Cell 10:117–128

    Article  PubMed  CAS  Google Scholar 

  • Hansen JL, Moore PB, Steitz TA (2003) Structures of five antibiotics bound at the peptidyl transferase center of the large ribosomal subunit. J Mol Biol 330:1061–1075

    Article  PubMed  CAS  Google Scholar 

  • Himeno H, Hanawa-Suetsugu K, Kimura T, Takagi K, Sugiyama W, Shirata S, Mikami T, Odagiri F, Osanai Y, Wantanabe D, Goto S, Kalachnyuk L, Ushida C, Muto A (2004) A novel GTPase activated by the small subunit of ribosome. Nucleic Acids Res 32:5303–5309

    Article  PubMed  CAS  Google Scholar 

  • Inoue K, Alsina J, Chen J, Inouye M (2003) Suppression of defective ribosome assembly in a rbfA deletion mutant by overexpression of Era, an essential GTPase in Escherichia coli. Mol Microbiol 48:1005–1016

    Article  PubMed  CAS  Google Scholar 

  • Kaczanowska M, Ryden-Aulin M (2005) The YrdC protein––a putative ribosome maturation factor. Biochem Biophys Acta 1727:87–96

    PubMed  CAS  Google Scholar 

  • Kirsebom LA, Vittanen A, Mikkelsen NE (2006) Aminoglycoside interactions with RNAs and nucleases. Hoppe Seiler Physiol Chem 173:75–96

    Google Scholar 

  • Kotra LP, Haddad J, Mobashery S (2000) Aminoglycosides: perspectives on mechanisms of action and resistance and strategies to counter resistance. Antimicrob Agents Chemother 44:3249–3256

    Article  PubMed  CAS  Google Scholar 

  • Lando D, Cousin A-M, Raynaud J-P (1976) Paromomycin and dihydrostreptomycin binding to Escherichia coli ribosomes. Eur J Biochem 66:597–606

    Article  PubMed  CAS  Google Scholar 

  • McGaha SM, Champney WS (2006) Hygromycin B inhibition of protein synthesis and ribosome biogenesis in Escherichia coli. Antimicrob Agents Chemother 51:591–596

    Article  PubMed  CAS  Google Scholar 

  • Mehta R, Champney WS (2002) 30S ribosomal subunit assembly is a target for inhibition by aminoglycosides in Escherichia coli. Antimicrob Agents Chemother 46:1546–1549

    Article  PubMed  CAS  Google Scholar 

  • Mehta R, Champney WS (2003) Neomycin and paromomycin inhibit 30S ribosomal subunit assembly in Staphylococcus aureus. Curr Microbiol 43:237–243

    Article  CAS  Google Scholar 

  • Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor

    Google Scholar 

  • Mingeot-LeClercq MP, Glupczynski Y, Tulkens PM (1999) Aminoglycosides: activity and resistance. Antimicrob Agents Chemother 43:727–737

    PubMed  CAS  Google Scholar 

  • Misumi M, Nishimura T, Komai T, Tanaka N (1978) Interaction of kanamycin and related antibiotics with the large subunit of ribosomes and the inhibition of translocation. Biochem Biophys Res Commun 84:358–365

    Article  PubMed  CAS  Google Scholar 

  • Moore PB (2001) The ribosome at atomic resolution. Biochemistry 40:3243–3250

    Article  PubMed  CAS  Google Scholar 

  • Nierhaus KH (1990) Reconstitution of ribosomes. In: Spedding G (eds) Ribosomes and protein synthesis. A practical approach. Oxford University Press, Oxford, pp 169–179

    Google Scholar 

  • Nierhaus K, Bordash K, Homann HE (1973) Ribosomal proteins. In vivo assembly of Escherichia coli ribosomal proteins. J Mol Biol 74:587–597

    Article  PubMed  CAS  Google Scholar 

  • Nomura M (1973) Assembly of bacterial ribosomes. Science 179:863–873

    Article  Google Scholar 

  • Ogle JM, Broderson DE, Clemons WM, Terry MJ, Carter AP, Ramakrishnan V (2001) Recognition of cognate transfer RNA by the 30S ribosomal subunit. Nature 292:897–902

    CAS  Google Scholar 

  • Ogle JM, Murphy FV, Tarry MJ, Ramakrishnan V (2002) Selection of tRNA by the ribosome requires a transition from an open to a closed form. Cell 111:721–732

    Article  PubMed  CAS  Google Scholar 

  • Ogle JM, Carter AP, Ramakrishnan V (2003) Insights into the decoding mechanism from recent ribosome structures. TIBS 28:259–266

    PubMed  CAS  Google Scholar 

  • Pfister P, Hobbie S, Brull C, Corti N, Vasella A, Westhof E, Bottger EC (2005) Mutagenesis of 16S rRNA C1409-G1491 base-pair differentiates between 6’OH and 6’NH3 aminoglycosides. J Mol Biol 346:467–475

    Article  PubMed  CAS  Google Scholar 

  • Purohit P, Stern S (1994) Interactions of a small RNA with antibiotic and RNA ligands of the 30S subunit. Nature 370:659–662

    Article  PubMed  CAS  Google Scholar 

  • Recht MI, Douthwaite S, Dahlquist KD, Puglisi JD (1999a) Effect of mutations in the A site of 16S RNA on aminoglycoside antibiotic-ribosome interactions. J Mol Biol 286:33–43

    Article  PubMed  CAS  Google Scholar 

  • Recht MI, Douthwaite S, Puglisi JD (1999b) Basis for prokaryotic specificity of action of aminoglycoside antibiotics. EMBO J 18:3133–3138

    Article  PubMed  CAS  Google Scholar 

  • Roets E, Adams E, Murithi I, Hoogmartens J (1995) Determination of the relative amounts of the B and C components of neomycin by thin-layer chromatography using fluorescence detection. J Chromatogr 696:131–138

    Article  CAS  Google Scholar 

  • Schlunzen F, Zarivach R, Harms J, Bashan A, Tocilj A, Albrecht R, Yonath A, Franceschi F (2001) Structural basis for the interaction of antibiotics with the peptidyltransferase centre in eubacteria. Nature 413:814–821

    Article  PubMed  CAS  Google Scholar 

  • Silvers JA, Champney WS (2005) Accumulation and turnover of 23S ribosomal RNA in azithromycin-inhibited ribonuclease mutants of Escherichia coli. Arch Mikrobiol 184:66–77

    CAS  Google Scholar 

  • Tor Y (2006) The ribosomal A-site as an inspiration for the design of RNA binders. Biochimie 88:1045–1051

    Article  PubMed  CAS  Google Scholar 

  • Toutchstone J, Dobbins M (1983) Practice of thin layer chromatography, 2nd edn. Wiley-Interscience, New York

    Google Scholar 

  • Usary J, Champney WS (2001) Erythromycin inhibition of 50S ribosomal subunit formation in Escherichia coli cells. Mol Microbiol 40:951–962

    Article  PubMed  CAS  Google Scholar 

  • Vazquez D (1979) Inhibitors of protein biosynthesis. Springer, Berlin

    Google Scholar 

  • Vourloumis D, Takahashi M, Winters GC, Simonsen KB, Ayida BK, Barluenga S, Qamar S, Shandrick S, Zhao Q, Hermann T (2002) Novel 2,5-dideoxystreptamine derivatives targeting the ribosomal decoding site RNA. Bioorg Med Chem Lett 12:3367–3372

    Article  PubMed  CAS  Google Scholar 

  • Zhou Y, Vlad E, Sun Z, Ayida B, Winters G, Murphy D, Simonsen K, Vourloumis D, Fish S, Froelich J, Wall D, Hermann T (2005) Structure-guided discovery of novel aminoglycoside mimetics as antibacterial translation inhibitors. Antimicrob Agents Chemother 49:4942–4949

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by a NIH Ruth L. Kirkchstein Predoctoral Fellowship and a NIH AREA grant.

Author information

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, J.H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN, 37614, USA

    Cerrone Foster & W. Scott Champney

Authors
  1. Cerrone Foster
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. W. Scott Champney
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to W. Scott Champney.

Additional information

Communicated by E. Stackebrandt.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Foster, C., Champney, W.S. Characterization of a 30S ribosomal subunit assembly intermediate found in Escherichia coli cells growing with neomycin or paromomycin. Arch Microbiol 189, 441–449 (2008). https://doi.org/10.1007/s00203-007-0334-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00203-007-0334-6

Keywords

Search

Navigation