Brandi Letizia, Marzi Stefano, Fabbretti Attilio, Fleischer Carola, Hill Walter E, Gualerzi Claudio O, Stephen Lodmell J
Laboratory of Genetics, Department of Biology MCA, University of Camerino, 62032 Camerino (MC), Italy.
J Mol Biol. 2004 Jan 23;335(4):881-94. 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.
利用游离的Cu(II):1,10 - 邻菲罗啉通过rRNA切割将细菌翻译起始因子IF2定位在核糖体上。结果表明IF2靠近螺旋89、帚曲霉素 - 蓖麻毒素环以及螺旋43和44，它们构成了23S rRNA的“L11/硫链丝菌素”茎环。这些发现促使人们研究L11对IF2活性的贡献，并重新审视关于硫链丝菌素对IF2功能影响这一有争议的问题，硫链丝菌素是一种主要作为转位强力抑制剂的肽类抗生素。发现缺乏L11的核糖体具有结合IF2的野生型能力，但引发其GTPase活性的能力大幅降低。我们发现硫链丝菌素导致该因子在70S核糖体和50S亚基上的结合与解离循环加快，这进而导致依赖IF2的GTPase多轮周转速率增加。尽管硫链丝菌素不抑制fMet - tRNA在P位点的结合、EF - Tu - GTP - Phe - tRNA在A位点的结合或核糖体肽基转移酶中心的活性（通过fMet - 嘌呤霉素的形成来衡量），但它严重抑制依赖IF2的起始二肽形成。这种抑制可能可追溯到硫链丝菌素诱导的IF2核糖体结合位点的扭曲，这导致核糖体与肽基转移酶反应的氨酰 - tRNA底物之间发生无效相互作用。总体而言，我们的数据表明IF2的翻译起始功能与EF - G的转位功能一样对硫链丝菌素抑制敏感。
