Structural characterization of the eukaryotic translation initiation by electron cryo-microscopy.

Translation is a highly conserved molecular process during which the genetic information stored in an mRNA molecule is translated into a polypeptide chain that eventually folds into functional three-dimensional proteins. Translation initiation, the first of four steps, is extremely important for the fidelity of the translation process. In eukaryotic translation initiation, the mRNA is brought into contact with the smaller 40S ribosomal subunit and subsequently scanned for the AUG start codon. Once identified, the mRNA gets locked in the P-site of the 40S ribosomal subunit via a constriction of its decoding center and a codon-anticodon-binding between the initiator Met-tRNAi and the start codon. The entire process is orchestrated and monitored by a large number of highly specialized factors that eventually trigger the joining of the larger 60S ribosomal subunit, followed by elongation of the polypeptide chain. Translation initiation has been the subject of extensively biochemical studies for many years. However, high-resolution reconstructions of the macromolecular complexes in- volved have just been obtained during the past decade. Thus, structural knowledge for this highly dynamic process is currently limited to a small number of complexes, depicting only narrow snapshots of what is happening. Additionally, although translation initiation is a highly conserved process, the factors involved vary quite extensively among eukary- otes. As a result, a comprehensive understanding of the dynamics and intermediate steps occurring during translation initiation, especially in higher mammals such as humans, remains missing. Here, two complexes involved in human translation initiation were structurally investi- gated: the free eukaryotic translation initiation factor 3 (eIF3) and the 48S initiation complex (48S-IC). eIF3, often described as a scaffold protein for all other initiation fac- tors and the 40S ribosomal subunit, is the largest of the initiation factors and is composed of 13 subunits. The 48S-IC is a late-stage initiation complex, in which the mRNA has already bound to the 40S together with many other initiation factors, including eIF3, to facilitate start codon recognition. The complex marks the final step before joining of the 60S ribosomal subunit and the start of translation elongation. For the free eIF3, a native purification procedure from HeLa cytoplasmic extract was established and the quality of the purified macromolecules has been validated via tandem mass spectrometry, ProteoPlex complex stability measurements, and protein crystalliza- tion. The purified eIF3 complexes were used to obtain a 3D reconstruction at 7–15 A resolution that exhibited a great degree of flexibility. Due to the fact that all 13 subunits of the complex have been identified by mass spectrometry and that the complex shows good structural integrity in other biochemical assays in particular ProteoPlex and protein crystallization it can be assumed that the flexibility is not a product of inappropriate sample preparation. Although it has not been possible thus far to stabilize the complexes enough to yield a high-resolution reconstruction for all of its parts, it was possible to ap- ply focused 3D classification to obtain several 3D structures that each show significantly more isotropic density than any other published eIF3 structure before. In order to study the interaction between the 40S ribosomal subunit and the eIF3 initiation factor, and additionally stabilize the composition of eIF3, the 48S-IC was investigated. Here, a large dataset was acquired which made it possible to extensively classify for structural heterogeneity and eventually yield a 3D reconstruction at 4.5–12 A resolution. It was possible to identify most of the initiation complexes attached to the surface of the 40S ribosomal subunit and verify their positions via cross-linking mass spectrometry. Especially for eIF1, an initiation factor involved in start codon recognition fidelity, it became possible to follow its track along several positions in the vicinity of the decoding center. In addition, the overall dynamics of the entire complex were studied using a three- dimensional principle component analysis-based approach. From these results, an energy landscape was calculated that revealed the tilting of the 40S head to be one of the major movements within in the complex with great impact on the stability. This movement occurs to hold the mRNA in the correct position once the start codon is identified. For the closed 40S head, significantly less structural flexibility was detected. Furthermore, movements in the surrounding of the 40S decoding center were also identified as one of the major sources of for structural heterogeneity. These results give a first insight into the important dynamics underlying the processes of eukaryotic translation and provide a promising foundation for future studies.