Department of Chemistry, Western Washington University, 516 High Street, MS 9150, Bellingham, WA 98225-9150, USA.
Nucleic Acids Res. 2012 Jan;40(1):360-70. doi: 10.1093/nar/gkr623. Epub 2011 Sep 9.
Thiostrepton, a macrocyclic thiopeptide antibiotic, inhibits prokaryotic translation by interfering with the function of elongation factor G (EF-G). Here, we have used 70S ribosome binding and GTP hydrolysis assays to study the effects of thiostrepton on EF-G and a newly described translation factor, elongation factor 4 (EF4). In the presence of thiostrepton, ribosome-dependent GTP hydrolysis is inhibited for both EF-G and EF4, with IC(50) values equivalent to the 70S ribosome concentration (0.15 µM). Further studies indicate the mode of thiostrepton inhibition is to abrogate the stable binding of EF-G and EF4 to the 70S ribosome. In support of this model, an EF-G truncation variant that does not possess domains IV and V was shown to possess ribosome-dependent GTP hydrolysis activity that was not affected by the presence of thiostrepton (>100 µM). Lastly, chemical footprinting was employed to examine the nature of ribosome interaction and tRNA movements associated with EF4. In the presence of non-hydrolyzable GTP, EF4 showed chemical protections similar to EF-G and stabilized a ratcheted state of the 70S ribosome. These data support the model that thiostrepton inhibits stable GTPase binding to 70S ribosomal complexes, and a model for the first step of EF4-catalyzed reverse-translocation is presented.
硫链丝菌素是一种大环硫肽抗生素，通过干扰延伸因子 G（EF-G）的功能来抑制原核翻译。在这里，我们使用 70S 核糖体结合和 GTP 水解测定法研究了硫链丝菌素对 EF-G 和新描述的翻译因子 EF4 的影响。在硫链丝菌素存在的情况下，核糖体依赖性 GTP 水解被 EF-G 和 EF4 抑制，IC50 值相当于 70S 核糖体浓度（0.15 µM）。进一步的研究表明，硫链丝菌素抑制的模式是使 EF-G 和 EF4 与 70S 核糖体的稳定结合丧失。支持该模型，一种不具有结构域 IV 和 V 的 EF-G 截断变体被证明具有核糖体依赖性 GTP 水解活性，不受硫链丝菌素的影响（>100 µM）。最后，化学足迹被用来检查与 EF4 相关的核糖体相互作用和 tRNA 运动的性质。在非水解 GTP 的存在下，EF4 显示出类似于 EF-G 的化学保护，并稳定了 70S 核糖体的棘轮状态。这些数据支持了硫链丝菌素抑制稳定的 GTPase 与 70S 核糖体复合物结合的模型，并提出了 EF4 催化反向易位的第一步模型。
