The translation initiation functions of IF2: targets for thiostrepton inhibition.
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.
Brandi
Letizia
L
Laboratory of Genetics, Department of Biology MCA, University of Camerino, 62032 Camerino (MC), Italy.
Marzi
Stefano
S
Fabbretti
Attilio
A
Fleischer
Carola
C
Hill
Walter E
WE
Gualerzi
Claudio O
CO
Stephen Lodmell
J
J
D001665:Binding Sites; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D020558:GTP Phosphohydrolases / Q000737:chemistry / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D008958:Models, Molecular; D010442:Peptide Chain Initiation, Translational / Q000187:drug effects*; D039881:Prokaryotic Initiation Factor-2 / Q000819:agonists / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D011487:Protein Conformation; D012343:RNA, Transfer / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism; D012269:Ribosomal Proteins / Q000378:metabolism; D012270:Ribosomes / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D013883:Thiostrepton / Q000494:pharmacology*
D039881:Prokaryotic Initiation Factor-2; D012269:Ribosomal Proteins; C027624:ribosomal protein L11; D006160:Guanosine Triphosphate; D012343:RNA, Transfer; D012260:Ribonucleases; D020558:GTP Phosphohydrolases; D013883:Thiostrepton