Structural and functional studies of eukaryotic translation initiation factors

Protein synthesis can be divided into three distinct subprocesses, namely initiation, elongation and termination. During the initiation phase, the elongation competent ribosome, which harbors the initiator tRNA in its P-site base-paired with the start codon of the mRNA, is assembled. Translation initiation is the most regulated and rate-limiting step. In eukaryotes, at least 12 distinct translation initiation factors (eIFs) facilitate this critical process. The largest among these factors is the multi-subunit complex eIF3 (6 subunits in yeast, 13 subunits (a-m) in mammals). eIF3 is involved in nearly all steps of the initiation process. However, the structure of eIF3 and the mechanism of its versatile functions are still largely elusive. In this work, the functional eIF3 core that is formed by 5 universally conserved subunits (a, b, c, g, and i) was reconstituted in vitro, and the interaction network was further explored. Within the 5-subunit eIF3 core, the b subunit (eIF3b) is shown to serve as the major scaffold. The crystal structure of the WD40 domain of Chaetomium thermophilum eIF3b was solved de novo by means of MAD. The structure revealed an unexpected nine-bladed WD40 β-propeller fold. It possesses a central channel that exhibits a circular truncated cone shape rather than the common cylindrical form. Sequence analysis suggests that this nine-bladed propeller architecture is adopted by all eIF3b orthologs. Based on its unique shape, the WD40 β-propeller of eIF3b was unambiguously fitted into the recently published cryo-EM map of the 43S preinitiation complex (PIC) (EMDB code: 5658). Due to this ribosomal location, underneath the shoulder of the 40S subunit, eIF3b is indicated to directly interact with the 40S ribosomal subunit. Consistently, stable interactions between eIF3b and the isolated ribosomal protein S9e, as well as the 40S subunit were observed using in vitro binding assays. Hence, these results strongly indicate a direct involvement of eIF3b in the assembly of the 43S PIC. The initiator tRNA bound 43S PIC is recruited to the 5’ untranslated region of the mRNA in a manner facilitated by eIF4F. eIF4F is a heterotrimeric complex, consisting of the m7G cap-binding protein eIF4E, the ATP-dependent RNA helicase eIF4A and the multi-scaffold eIF4G. eIF4A melts secondary structures in the 5’ UTR for recruitment of the 43S PIC and subsequent scanning. However, the helicase and ATPase activities of isolated eIF4A are very low. High activities occur only when eIF4B and eIF4G are present. In this thesis, a potential RNA-binding fragment of eIF4G is shown to be indispensable for the efficient stimulation. The RNA binding ability and ATPase activity of eIF4A was dramatically increased in the presence of an eIF4G variant that contains eIF4A- and RNA-binding regions. By contrast, an eIF4G truncation containing the eIF4A-binding domain only did not show any enzymatic enhancement. These findings suggest that eIF4G enhances the activities of eIF4A through simultaneous interactions with eIF4A and RNA, and providing the RNA substrate for eIF4A. This model was further supported by site-specific crosslinking experiments