Nature. 2016 Jul 28;535(7613):570-4. doi: 10.1038/nature18647. Epub 2016 Jul 20.
Regulation of messenger RNA translation is central to eukaryotic gene expression control. Regulatory inputs are specified by them RNA untranslated regions (UTRs) and often target translation initiation. Initiation involves binding of the 40S ribosomal small subunit (SSU) and associated eukaryotic initiation factors (eIFs)near the mRNA 5′ cap; the SSU then scans in the 3′ direction until it detects the start codon and is joined by the 60S ribosomal large subunit (LSU) to form the 80S ribosome. Scanning and other dynamic aspects of the initiation model have remained as conjectures because methods to trap early intermediates were lacking. Here we uncover the dynamics of the complete translation cycle in live yeast cells using translation complex profile sequencing (TCP-seq), a method developed from the ribosome profiling approach. We document scanning by observing SSU footprints along 5′ UTRs. Scanning SSU have 5′-extended footprints (up to~75 nucleotides), indicative of additional interactions with mRNA emerging from the exit channel, promoting forward movement. We visualized changes in initiation complex conformation as SSU footprints coalesced into three major sizes at start codons (19, 29 and 37 nucleotides). These share the same 5′ start site but differ at the 3′ end, reflecting successive changes at the entry channel from an open to a closed state following start codon recognition. We also observe SSU 'lingering' at stop codons after LSU departure. Our results underpin mechanistic models of translation initiation and termination, built on decades of biochemical and structural investigation, with direct genome-wide in vivo evidence. Our approach captures ribosomal complexes at all phases of translation and will aid in studying translation dynamics in diverse cellular contexts. Dysregulation of translation is common in disease and, for example, SSU scanning is a target of anti-cancer drug development. TCP-seq will prove useful in discerning differences in mRNA-specific initiation in pathologies and their response to treatment.
mRNA 翻译的调控是真核生物基因表达调控的核心。调节输入由 RNA 非翻译区 (UTR) 指定，通常靶向翻译起始。起始涉及到 40S 核糖体小亚基 (SSU) 和相关的真核起始因子 (eIF) 在 mRNA 5'帽附近的结合；然后，SSU 从 3'方向扫描，直到它检测到起始密码子，并与 60S 核糖体大亚基 (LSU) 结合形成 80S 核糖体。由于缺乏捕获早期中间产物的方法，扫描和起始模型的其他动态方面仍然是推测。在这里，我们使用翻译复合物图谱测序 (TCP-seq) 方法，该方法源自核糖体图谱方法，在活酵母细胞中揭示了完整翻译周期的动态。我们通过观察 5'UTR 上的 SSU 足迹来记录扫描。扫描 SSU 具有 5'延伸的足迹（最多~75 个核苷酸），表明与从出口通道中出现的 mRNA 有额外的相互作用，促进了向前运动。我们观察到起始复合物构象的变化，因为 SSU 足迹在起始密码子处合并成三个主要大小（19、29 和 37 个核苷酸）。这些足迹具有相同的 5'起始位点，但在 3'端不同，反映了在起始密码子识别后，入口通道从开放状态到关闭状态的连续变化。我们还观察到 LSU 离开后 SSU 在终止密码子处“停留”。我们的结果为基于数十年生化和结构研究建立的翻译起始和终止的机制模型提供了依据，并提供了直接的全基因组体内证据。我们的方法可以捕获翻译的所有阶段的核糖体复合物，并有助于在不同的细胞环境中研究翻译动态。翻译的失调在疾病中很常见，例如，SSU 扫描是抗癌药物开发的靶点。TCP-seq 将有助于辨别病理学中 mRNA 特异性起始的差异及其对治疗的反应。
