Eukaryotic Translation Initiation Factor Eif3 And Eif5 Structural Biology

Eukaryotic translation initiation is a sophisticated cellular process, which is stimulated by a number of proteins called eukaryotic initiation factors (eIFs). At least 12 eIFs have been identified. Using protein crystallography, we solved the eIF3k and eIF5-CTD structures; and we also employed electron microscopy to analyze the eIF3 complex structure. By studying the structure-function relationships of eIF3 and eIF5, we provided key information for understanding protein biosynthesis in eukaryotes.eIF3 is the largest and most complicated initiation factor, which plays a central role in the translation initiation process. Mammalian eIF3 contains 12 subunits, among which eIF3k is the smallest. eIF3k is conserved through high eukaryotes, including mammals, insects, and plants. However, eIF3k does not exist in some species of yeast, suggesting that eIF3k may play a unique regulatory role in higher eukaryotes. Human eIF3k structure is the first high-resolution structure of an eIF3 component. This novel structure contains two distinct domains, a HEAT repeat-like HAM (HEAT analogous motif) domain and a WH (winged-helix) domain. The HAM domain is a super-helical motif, while the WH domain belongs to helix-turn-helix motif. Although most WH domains are found to interact with DNA or RNA, the WH domain of human eIF3k is probably involved in protein-protein interactions based on carefully structural analysis. In addition, HEAT motifs are also involved in protein-protein interaction. Taking together, we conclude that eIF3k should be a scaffold protein. By analyzing the conserved residue distribution on protein surface, we proposed three potential binding surfaces for other eIF3 subunits. In order to discover the whole structure of eIF3 further, we prepared the eIF3 complex sample and obtained preliminary structural information by electron microscopy.eIF5, a GTPase-activating protein specific for eIF2, plays a critical role in pre-initiation complex assembly and ensures correct AUG selection during eukaryotic translation initiation. eIF5 is involved in the formation of the multifactor complex (MFC), an important intermediate of the 43 S preinitiation complex. By interacting with eIF2 and eIF3, the eIF5 C-terminal domain (eIF5-CTD) functions as a structural core in the MFC assembly, which indicates that eIF5-CTD also acts as a scaffold protein. Our structure shows that eIF5-CTD contains an atypical HEAT motif. By analyzing the electrostatic potential and the distribution of conserved residues on the protein surface, we confirmed and meanwhile proposed some potential regions of interactions between eIF5-CTD and other eIFs. The structure of eIF5-CTD provides useful information in understanding the mechanism of MFC assembly.Eukaryotic translation initiation is a complicated system, composed of numerous protein-protein interactions. In eIFs, there are many scaffold proteins, which act as structural cores in the complex system by connecting related eIFs together. Including eIF3k and eIF5-CTD, many scaffold proteins with 3D structure in eIFs contain HEAT or HEAT-like motifs. The phenomenon indicates that HEAT motifs are most extremely structural units in eukaryotic translation initiation.