During translation initiation, mRNA molecules must be identified and activated for loading into a ribosome^1,2,3. In this rate-limiting step, the heterotrimeric protein eukaryotic initiation factor eIF4F must recognize and productively interact with the 7-methylguanosine cap at the 5′ end of the mRNA and subsequently activate the message^1,2,3. Despite its fundamental, regulatory role in gene expression, the molecular events underlying cap recognition and mRNA activation remain unclear³. Here we generate a single-molecule fluorescence imaging system to examine the dynamics with which eIF4F discriminates productive and non-productive locations on full-length, native mRNA molecules. At the single-molecule level, we observe stochastic sampling of eIF4F along the length of the mRNA and identify allosteric communication between the eIF4F subunits that ultimately drive cap-recognition and subsequent activation of the message. Our experiments uncover functions for each subunit of eIF4F and we conclude by presenting a model for mRNA activation that precisely defines the composition of the activated message. This model provides a general framework for understanding how mRNA molecules may be discriminated from one another and how other RNA-binding proteins may control the efficiency of translation initiation.

在翻译起始过程中，必须识别并激活 mRNA 分子以加载到核糖体中^1,2,3。在这个限速步骤中，异源三聚体蛋白真核起始因子 eIF4F 必须识别 mRNA 5' 端的 7-甲基鸟苷帽并与之有效相互作用，随后激活信息^1,2,3。尽管它在基因表达中具有基本的调节作用，但 cap 识别和 mRNA 激活的分子事件仍不清楚³。在这里，我们生成了一个单分子荧光成像系统，以检查 eIF4F 区分全长天然 mRNA 分子上的生产性和非生产性位置的动力学。在单分子水平上，我们观察到 eIF4F 沿 mRNA 长度的随机采样，并确定了 eIF4F 亚基之间的变构通讯，这些亚基最终驱动帽识别和随后的信息激活。我们的实验揭示了 eIF4F 每个亚基的功能，最后我们提出了一个 mRNA 激活模型，该模型精确定义了激活信息的组成。该模型为理解 mRNA 分子如何相互区分以及其他 RNA 结合蛋白如何控制翻译起始的效率提供了一个通用框架。