Dynamics of ribosome scanning and recycling revealed by translation complex profiling
Dynamics of ribosome scanning and recycling revealed by translation complex profiling
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DOI：
10.1038/nature18647
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发表时间：
2016-07-28
期刊：
影响因子：
64.8
通讯作者：
Preiss, Thomas
中科院分区：
文献类型：
--
作者：
Archer, Stuart K.;Shirokikh, Nikolay E.;Preiss, Thomas
信使RNA翻译的调节是真核基因表达控制的核心（1）。调控输入由mRNA非翻译区（UTR）指定，并且通常靶向翻译起始。起始涉及40 S核糖体小亚基（SSU）和mRNA 5'帽附近的相关真核起始因子（eIF）的结合;然后SSU沿3'方向扫描，直至检测到起始密码子，并与60 S核糖体大亚基（LSU）连接（2-5），形成80 S核糖体。由于缺乏捕获早期中间体的方法，引发模型的扫描和其他动力学方面仍然是一个难题。在这里，我们使用翻译复合物谱测序（TCP-seq）揭示了活酵母细胞中完整翻译周期的动态，TCP-seq是一种从核糖体谱（6）方法开发的方法。我们通过沿沿着5'UTR观察SSU足迹来记录扫描。扫描SSU具有5 '-延伸的足迹（高达类似于75个核苷酸），指示与从出口通道出现的mRNA的额外相互作用，促进向前移动。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（19、29和37个核苷酸）处合并成三个主要尺寸。这些共有相同的5'起始位点，但在3'末端不同，反映了起始密码子识别后进入通道从开放状态到闭合状态的连续变化。我们还观察到SSU在LSU离开后在停止密码子处“徘徊”。我们的研究结果支持翻译起始和终止的机制模型，建立在数十年的生化和结构研究的基础上，具有直接的全基因组体内证据。我们的方法捕获在翻译的所有阶段的核糖体复合物，并将有助于在不同的细胞环境中研究翻译动力学。翻译失调在疾病中很常见，例如，SSU扫描是抗癌药物开发的目标（7）。TCP-seq将被证明可用于辨别病理学中mRNA特异性起始及其对治疗的反应的差异。
Regulation of messenger RNA translation is central to eukaryotic gene expression control(1). Regulatory inputs are specified by the mRNA 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)(2-5) 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(6) approach. We document scanning by observing SSU footprints along 5' UTRs. Scanning SSU have 5'-extended footprints (up to similar 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(7). TCP-seq will prove useful in discerning differences in mRNA-specific initiation in pathologies and their response to treatment.