Abstract
The eukaryotic translation initiation is a biological process in which at least a dozen eukaryotic initiation factors (eIFs) are involved. Specifically, eIF3, eIF1, eIF5, and eIF2 as a ternary complex (eIF2-TC) bound to GTP and methionyl initiator tRNA (Met-tRNA(i) (Met)). They interact to form a large complex called the multifactor complex (MFC). This complex binds cooperatively to the ribosomal pre-initiation complex (PIC), promoting the loading of the Met-tRNA(i) (Met) into the peptidyl (P) site of the 40S ribosomal subunit. While some interactions between eIFs have been described in the context of the PIC, the interactions within the MFC remain poorly understood. Here, we combine biophysical and biochemical approaches, including mass photometry and native mass spectrometry, with structural biology methods such as electron microscopy, to gain deeper insights into the MFC architecture. Our findings provide novel insights into the critical role of eIF5 during MFC assembly. Notably, two copies of eIF5 are involved in the formation of the MFC. We propose that one eIF5 molecule engages eIF2β and eIF2γ, whereas a second eIF5 molecule interacts with eIF1 together with eIF3c.