Translation initiation controls protein synthesis by regulating the delivery of the first aminoacyl-tRNA to messenger RNAs (mRNAs). In eukaryotes, initiation is sophisticated, requiring dozens of protein factors and 2 GTP-regulated steps. The GTPase eIF5B gates progression to elongation during the second GTP-regulated step. Using electron cryomicroscopy (cryo-EM), we imaged an in vitro initiation reaction which is set up with purified yeast components and designed to stall with eIF5B and a nonhydrolyzable GTP analog. A high-resolution reconstruction of a "dead-end" intermediate at 3.6 Å allowed us to visualize eIF5B in its ribosome-bound conformation. We identified a stretch of residues in eIF5B, located close to the γ-phosphate of GTP and centered around the universally conserved tyrosine 837 (Saccharomyces cerevisiae numbering), that contacts the catalytic histidine of eIF5B (H480). Site-directed mutagenesis confirmed the essential role that these residues play in regulating ribosome binding, GTP hydrolysis, and translation initiation both in vitro and in vivo. Our results illustrate how eIF5B transmits the presence of a properly delivered initiator aminoacyl-tRNA at the P site to the distant GTPase center through interdomain communications and underscore the importance of the multidomain architecture in translation factors to sense and communicate ribosomal states.

中文翻译：

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eIF5B中的远程域间通信调节GTP水解和翻译起始。

翻译起始通过调节第一个氨酰基-tRNA到信使RNA（mRNA）的传递来控制蛋白质的合成。在真核生物中，起始是复杂的，需要数十种蛋白质因子和2个GTP调控的步骤。在第二个GTP调节步骤中，GTPase eIF5B门控延伸至延伸。使用电子冷冻显微镜（cryo-EM），我们对体外起始反应进行了成像，该反应是用纯化的酵母成分建立的，旨在与eIF5B和不可水解的GTP类似物停滞。在3.6埃处“末端”中间体的高分辨率重建，使我们可以看到eIF5B的核糖体结合构象。我们在eIF5B中鉴定了一段残基，其残基靠近GTP的γ-磷酸，并以普遍保守的酪氨酸837（酿酒酵母编号）为中心，与eIF5B（H480）的催化组氨酸接触。定点诱变证实了这些残基在体外和体内均在调节核糖体结合，GTP水解和翻译起始中起着至关重要的作用。我们的结果说明了eIF5B如何通过域间通信将P位点上正确递送的引发剂氨酰基tRNA的存在传递给遥远的GTPase中心，并强调了多域结构在翻译因子中检测和传递核糖体状态的重要性。