Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa-shi, Chiba 277-8562, Japan.
J Biochem. 2018 Mar 1;163(3):177-185. doi: 10.1093/jb/mvx070.
The stress-related protein Stm1 interacts with ribosomes, and is implicated in repressing translation. Stm1 was previously studied both in vivo and in vitro by cell-free translation systems using crude yeast lysates, but its precise functional mechanism remains obscure. Using an in vitro reconstituted translation system, we now show that Stm1 severely inhibits translation through its N-terminal region, aa 1 to 107, and this inhibition is antagonized by eEF3. We found that Stm1 stabilizes eEF2 on the 80 S ribosome in the GTP-bound form, independently of eEF2's diphthamide modification, a conserved post-translational modification at the tip of domain IV. Systematic analyses of N- or C-terminal truncated mutants revealed that the core region of Stm1, aa 47 to 143, is crucial for its ribosome binding and eEF2 stabilization. Stm1 does not inhibit the 80 S-dependent GTPase activity of eEF2, at least during the first round of GTP-hydrolysis. The mechanism and the role of the stable association of eEF2 with the ribosome in the presence of Stm1 are discussed in relation to the translation repression by Stm1.
应激相关蛋白 Stm1 与核糖体相互作用，并被认为在抑制翻译中起作用。以前曾使用粗酵母裂解物在无细胞翻译系统中对 Stm1 进行体内和体外研究，但它的确切功能机制仍不清楚。使用体外重组成套翻译系统，我们现在表明 Stm1 通过其 N 端区域（aa1 至 107）严重抑制翻译，这种抑制作用被 eEF3 拮抗。我们发现 Stm1 独立于 eEF2 的二氢尿嘧啶修饰，即结构域 IV 顶端的保守翻译后修饰，稳定地将 eEF2 稳定在 GTP 结合的 80S 核糖体上。对 N 端或 C 端截断突变体的系统分析表明，Stm1 的核心区域（aa47 至 143）对于其核糖体结合和 eEF2 稳定至关重要。Stm1 不会抑制 eEF2 的 80S 依赖性 GTPase 活性，至少在第一轮 GTP 水解过程中不会。讨论了在 Stm1 存在下稳定结合 eEF2 与核糖体的机制及其在 Stm1 抑制翻译中的作用。
