Murray Jason, Savva Christos G, Shin Byung-Sik, Dever Thomas E, Ramakrishnan V, Fernández Israel S
MRC Laboratory of Molecular Biology, Cambridge, United Kingdom.
Laboratory of Gene Regulation and Development, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, United States.
Elife. 2016 May 9;5:e13567. doi: 10.7554/eLife.13567.
Viral mRNA sequences with a type IV IRES are able to initiate translation without any host initiation factors. Initial recruitment of the small ribosomal subunit as well as two translocation steps before the first peptidyl transfer are essential for the initiation of translation by these mRNAs. Using electron cryomicroscopy (cryo-EM) we have structurally characterized at high resolution how the Cricket Paralysis Virus Internal Ribosomal Entry Site (CrPV-IRES) binds the small ribosomal subunit (40S) and the translocation intermediate stabilized by elongation factor 2 (eEF2). The CrPV-IRES restricts tvhe otherwise flexible 40S head to a conformation compatible with binding the large ribosomal subunit (60S). Once the 60S is recruited, the binary CrPV-IRES/80S complex oscillates between canonical and rotated states (Fernández et al., 2014; Koh et al., 2014), as seen for pre-translocation complexes with tRNAs. Elongation factor eEF2 with a GTP analog stabilizes the ribosome-IRES complex in a rotated state with an extra ~3 degrees of rotation. Key residues in domain IV of eEF2 interact with pseudoknot I (PKI) of the CrPV-IRES stabilizing it in a conformation reminiscent of a hybrid tRNA state. The structure explains how diphthamide, a eukaryotic and archaeal specific post-translational modification of a histidine residue of eEF2, is involved in translocation.
具有IV型内部核糖体进入位点（IRES）的病毒mRNA序列能够在没有任何宿主起始因子的情况下启动翻译。小核糖体亚基的初始招募以及第一个肽基转移之前的两个转位步骤对于这些mRNA启动翻译至关重要。利用低温电子显微镜（cryo-EM），我们在高分辨率下对蟋蟀麻痹病毒内部核糖体进入位点（CrPV-IRES）如何结合小核糖体亚基（40S）以及由延伸因子2（eEF2）稳定的转位中间体进行了结构表征。CrPV-IRES将原本灵活的40S头部限制在与结合大核糖体亚基（60S）兼容的构象中。一旦招募了60S，二元CrPV-IRES/80S复合物就在经典状态和旋转状态之间振荡（Fernández等人，2014年；Koh等人，2014年），就像带有tRNA的转位前复合物一样。带有GTP类似物的延伸因子eEF2将核糖体-IRES复合物稳定在旋转状态，额外旋转约3度。eEF2结构域IV中的关键残基与CrPV-IRES的假结I（PKI）相互作用，将其稳定在类似于杂合tRNA状态的构象中。该结构解释了二磷酸酰胺（一种对eEF2组氨酸残基进行的真核和古细菌特异性翻译后修饰）如何参与转位。
