The translation initiation functions of IF2: Targets for thiostrepton inhibition
The translation initiation functions of IF2: Targets for thiostrepton inhibition
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DOI：
10.1016/j.jmb.2003.10.067
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发表时间：
2004-01-23
影响因子：
5.6
通讯作者：
Lodmell, JS
中科院分区：
文献类型：
--
作者：
Brandi, L;Marzi, S;Lodmell, JS
利用游离铜(II)：1，10-邻菲咯啉对rRNA进行切割，将细菌翻译起始因子IF2定位在核糖体上。结果表明，IF2与螺旋、sarcin-ricin环、43和44螺旋距离较近，构成了23个S rRNA的“L11/硫链”茎环。这些发现促使了对L11对IF2活性的贡献的调查，并重新审视了硫链菌素对IF2功能的影响这一有争议的问题，硫链菌素是一种多肽抗生素，主要被认为是一种强大的易位抑制剂。缺乏L11的核糖体具有与IF2结合的野生型能力，但诱导其GTPase活性的能力显著降低。我们发现，硫链菌素导致该因子在70个S核糖体和50个S亚基上和外循环得更快，这反过来又导致依赖于IF2的GTP酶多次周转的速率增加。尽管硫链菌素不能抑制fMet-tRNA的P位结合、EF-Tu-GTP-Phe-tRNA的A位结合或核糖体转移酶中心的活性(用fMet-puromycin的形成来衡量)，但它严重地抑制了IF2依赖的起始二肽的形成。这种抑制可能可以追溯到硫链菌素诱导的1172位核糖体结合位点的扭曲，这导致了核糖体与肽基转移酶反应的氨基酰tRNA底物之间的非生产性相互作用。总体而言，我们的数据表明，IF2的翻译起始功能与EF-G的易位功能一样对硫链霉菌抑制敏感。(C)2003爱思唯尔有限公司。保留所有权利。
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 23 S 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 70 S ribosomes and 50 S 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 1172, 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. (C) 2003 Elsevier Ltd. All rights reserved.