The translation initiation functions of IF2: targets for thiostrepton inhibition

• Published in: Journal of molecular biology 2004-01, Vol.335 (4), p.881-894
• Main Authors: Brandi, Letizia, Marzi, Stefano, Fabbretti, Attilio, Fleischer, Carola, Hill, Walter E, Gualerzi, Claudio O, Stephen Lodmell, J
• Format: Article
• Language: English
• Subjects: Binding Sites; Enzyme Activation - drug effects; Escherichia coli - enzymology; Escherichia coli - genetics; GTP Phosphohydrolases - chemistry; GTP Phosphohydrolases - metabolism; Guanosine Triphosphate - metabolism; initiation factor eIF-2; initiation factor IF-2; Models, Molecular; Peptide Chain Initiation, Translational - drug effects; Prokaryotic Initiation Factor-2 - agonists; Prokaryotic Initiation Factor-2 - chemistry; Prokaryotic Initiation Factor-2 - metabolism; Protein Binding - drug effects; Protein Conformation; Ribonucleases - metabolism; Ribosomal Proteins - metabolism; Ribosomes - chemistry; Ribosomes - drug effects; Ribosomes - metabolism; RNA, Transfer - metabolism; Thiostrepton - pharmacology
• ISSN: 0022-2836
• DOI: 10.1016/j.jmb.2003.10.067

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.