Hussain Tanweer, Llácer Jose L, Fernández Israel S, Munoz Antonio, Martin-Marcos Pilar, Savva Christos G, Lorsch Jon R, Hinnebusch Alan G, Ramakrishnan V
MRC Laboratory of Molecular Biology, Cambridge CB2 0QH, UK.
Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Laboratory on the Mechanism and Regulation of Protein Synthesis, Eunice K. Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.
Cell. 2014 Oct 23;159(3):597-607. doi: 10.1016/j.cell.2014.10.001. Epub 2014 Oct 16.
During eukaryotic translation initiation, initiator tRNA does not insert fully into the P decoding site on the 40S ribosomal subunit. This conformation (POUT) is compatible with scanning mRNA for the AUG start codon. Base pairing with AUG is thought to promote isomerization to a more stable conformation (PIN) that arrests scanning and promotes dissociation of eIF1 from the 40S subunit. Here, we present a cryoEM reconstruction of a yeast preinitiation complex at 4.0 Å resolution with initiator tRNA in the PIN state, prior to eIF1 release. The structure reveals stabilization of the codon-anticodon duplex by the N-terminal tail of eIF1A, changes in the structure of eIF1 likely instrumental in its subsequent release, and changes in the conformation of eIF2. The mRNA traverses the entire mRNA cleft and makes connections to the regulatory domain of eIF2?, eIF1A, and ribosomal elements that allow recognition of context nucleotides surrounding the AUG codon.
在真核生物翻译起始过程中，起始tRNA不会完全插入40S核糖体亚基上的P解码位点。这种构象（POUT）与扫描mRNA寻找AUG起始密码子的过程是相容的。与AUG的碱基配对被认为会促进异构化，形成更稳定的构象（PIN），从而停止扫描并促进eIF1从40S亚基上解离。在这里，我们展示了一种酵母起始前复合物的冷冻电镜重建结构，分辨率为4.0 Å，其中起始tRNA处于PIN状态，且eIF1尚未释放。该结构揭示了eIF1A的N端尾巴对密码子-反密码子双链体的稳定作用、eIF1结构的变化（这可能对其随后的释放起作用）以及eIF2构象的变化。mRNA穿过整个mRNA裂隙，并与eIF2α、eIF1A的调节结构域以及核糖体元件建立连接，从而能够识别AUG密码子周围的上下文核苷酸。
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