Molecular Architecture of a Eukaryotic Translational Initiation Complex

作者：I.S. Fernandez, Xiao‐chen Bai, Tanweer Hussain, Ann C. Kelley, Jon R. Lorsch, V. Ramakrishnan, Sjors H. W. Scheres · 发表于：Science · 年份：2013 · DOI：10.1126/science.1240585 · 被引用次数：132 · 研究领域：RNA and protein synthesis mechanisms、CRISPR and Genetic Engineering、RNA modifications and cancer

Introduction Initiation of protein synthesis is a key step in the control of gene expression. In eukaryotes, initiation is a highly complex process that requires almost a dozen protein factors. The last step involves joining of the large and small subunits of the ribosome to form the 80 S initiation complex with the transfer RNA (tRNA) in the P-site base paired to the start codon. This step is catalyzed by the guanosine triphosphatase (GTPase) factor eIF5B. In addition, eIF5B is thought to play a role in ensuring that translation initiation takes place only on mature ribosomes. Methods The development of fast, direct electron detectors and new methods of image analysis for cryo–electron microscopy allow high-resolution reconstructions from much smaller numbers of particles than previously possible. We used these new methods to provide feedback to improve the biochemical preparation of samples for structure determination of the eukaryotic translation initiation complex with initiator tRNA and eIF5B, which was trapped on the ribosome with the nonhydrolyzable GTP analog GDPCP. Results Although the structure of the fully assembled complex was calculated from only 5143 particles, representing just 3% of the population in the sample, it was possible to obtain a resolution of 6.6 Å. This allowed us to propose a molecular model for the initiation complex. The structure shows that the subunits of the ribosome are rotated relative to the canonical state after initiation. The long helix...