The Translation Initiation Functions of IF2: Targets for Thiostrepton Inhibition

Letizia Brandi, Stefano Marzi, Attilio Fabbretti, Carola Fleischer, Walter E. Hill, Claudio O. Gualerzi, J. Stephen Lodmell

Research output: Contribution to journalArticlepeer-review

Abstract

Bacterial translation initiation factor IF2 was localized on the ribosome by rRNA cleavage using free Cu(II):1,10-orthophenanthroline. The results indicated proximity of IF2 to helix 89, to the sarcin-ricin loop and to helices 43 and 44, which constitute the "L11/thiostrepton" stem-loops of 23S rRNA. These findings prompted an investigation of the L11 contribution to IF2 activity and a re-examination of the controversial issue of the effect on IF2 functions of thiostrepton, a peptide antibiotic known primarily as a powerful inhibitor of translocation. Ribosomes lacking L11 were found to have wild-type capacity to bind IF2 but a strongly reduced ability to elicit its GTPase activity. We found that thiostrepton caused a faster recycling of this factor on and off the 70S ribosomes and 50S subunits, which in turn resulted in an increased rate of the multiple turnover IF2-dependent GTPase. Although thiostrepton did not inhibit the P-site binding of fMet-tRNA, the A-site binding of the EF-Tu-GTP-Phe-tRNA or the activity of the ribosomal peptidyl transferase center (as measured by the formation of fMet-puromycin), it severely inhibited IF2-dependent initiation dipeptide formation. This inhibition can probably be traced back to a thiostrepton-induced distortion of the ribosomal-binding site of IF2, which leads to a non-productive interaction between the ribosome and the aminoacyl-tRNA substrates of the peptidyl transferase reaction. Overall, our data indicate that the translation initiation function of IF2 is as sensitive as the translocation function of EF-G to thiostrepton inhibition.

Original languageEnglish
Pages (from-to)881-894
Number of pages14
JournalJournal of Molecular Biology
Volume335
Issue number4
DOIs
StatePublished - Jan 23 2004

Keywords

  • Antibiotic inhibition
  • Chemical Rnase
  • Ribosomal protein L11
  • Ribosomal topography
  • Translation initiation

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