A recent crystal structure of a ribosome complex undergoing partial translocation in the absence of elongation factor EF-G showed disruption of codon-anticodon pairing and slippage of the reading frame by -1, directly implicating EF-G in preservation of the translational reading frame. Among mutations identified in a random screen for dominant-lethal mutations of EF-G were a cluster of 6 that map to the tip of domain IV, which has been shown to contact the codon-anticodon duplex in trapped translocation intermediates. In vitro synthesis of a full-length protein using these mutant EF-Gs revealed dramatically increased -1 frameshifting, providing new evidence for a role for domain IV of EF-G in maintaining the reading frame. These mutations also caused decreased rates of mRNA translocation and rotational movement of the head and body domains of the 30S ribosomal subunit during translocation. Our results are in general agreement with recent findings from Rodnina and co-workers based on in vitro translation of an oligopeptide using EF-Gs containing mutations at two positions in domain IV, who found an inverse correlation between the degree of frameshifting and rates of translocation. Four of our six mutations are substitutions at positions that interact with the translocating tRNA, in each case contacting the RNA backbone of the anticodon loop. We suggest that EF-G helps to preserve the translational reading frame by preventing uncoupled movement of the tRNA through these contacts; a further possibility is that these interactions may stabilize a conformation of the anticodon that favors base-pairing with its codon.

中文翻译：

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延伸因子 EF-G 域 IV 中的突变赋予 -1 移码

在没有延伸因子 EF-G 的情况下进行部分易位的核糖体复合物的最新晶体结构显示密码子-反密码子配对的破坏和阅读框的滑动 -1，直接暗示 EF-G 保存了翻译阅读框。在 EF-G 显性致死突变的随机筛选中确定的突变中有 6 个簇，它们映射到结构域 IV 的尖端，已显示其与受困易位中间体中的密码子-反密码子双链体接触。使用这些突变 EF-G 体外合成全长蛋白质显示 -1 移码显着增加，为 EF-G 的域 IV 在维持阅读框架中的作用提供了新的证据。这些突变还导致易位期间 30S 核糖体亚基的头部和身体结构域的 mRNA 易位和旋转运动率降低。我们的结果与 Rodnina 和同事最近的发现大体一致，他们基于使用 EF-G 在结构域 IV 的两个位置包含突变的寡肽的体外翻译，他们发现移码程度和易位率之间存在负相关. 我们的六个突变中有四个是在与易位 tRNA 相互作用的位置上的替换，在每种情况下都与反密码子环的 RNA 骨架接触。我们建议 EF-G 通过防止 tRNA 通过这些接触的非耦合运动来帮助保留翻译阅读框架；