The mechanism for how internal ribosome entry sites (IRESs) recruit ribosomes to initiate translation of an mRNA is not completely understood. We investigated how a 40S subunit was recruited by the cricket paralysis virus intergenic region (CrPV IGR) IRES to form a stable 40S–IRES complex. Kinetic binding studies revealed that formation of the complex between the CrPV IGR and the 40S subunit consisted of two-steps: an initial fast binding step of the IRES to the 40S ribosomal subunit, followed by a slow unimolecular reaction consistent with a conformational change that stabilized the complex. We further showed that the ribosomal protein S25 (eS25), which is required by functionally and structurally diverse IRESs, impacts both steps of the complex formation. Mutations in eS25 that reduced CrPV IGR IRES activity either decreased 40S–IRES complex formation, or increased the rate of the conformational change that was required to form a stable 40S–IRES complex. Our data are consistent with a model in which eS25 facilitates initial binding of the CrPV IGR IRES to the 40S while ensuring that the conformational change stabilizing the 40S–IRES complex does not occur prematurely.

中文翻译：

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病毒IRES与40S亚基的结合在由eS25介导的两个连续步骤中发生。

内部核糖体进入位点（IRES）如何募集核糖体以启动mRNA翻译的机制尚不完全清楚。我们调查了how麻痹病毒基因间区域（CrPV IGR）IRES如何招募40S亚基，以形成稳定的40S-IRES复合物。动力学结合研究表明，CrPV IGR和40S亚基之间的复合物形成包括两个步骤：IRES最初与40S核糖体亚基快速结合的步骤，然后是缓慢的单分子反应，其构象变化稳定了复杂。我们进一步表明，功能和结构上不同的IRES需要核糖体蛋白S25（eS25），影响复合物形成的两个步骤。降低CrPV IGR IRES活性的eS25突变降低了40S-IRES复合物的形成，或增加了形成稳定的40S-IRES复合物所需的构象变化率。我们的数据与其中eS25促进CrPV IGR IRES与40S的初始结合并同时确保稳定40S-IRES复合物的构象变化不会过早发生的模型相符。