Biochemical and structural studies to provide insights into initiator tRNA delivery by eIF2A in noncanonical translation initiation

Abstract

Eukaryotic initiation factor 2A (eIF2A), a non-canonical translation initiation factor, delivers initiator tRNA (tRNAi) onto the 40S ribosomal subunit in a codon-dependent and GTP-independent manner, unlike the canonical initiation factor eIF2. eIF2A helps to perform the stress response under global protein synthesis inhibition by translating some specific mRNAs, hence regulating their protein synthesis. However, the underlying mechanism of this eIF2A-mediated non-canonical translation initiation is not known. It has a biologically significant role in various diseases like early tumorigenesis, neuromuscular disorders, viral infection, and Integrated stress response. Hence, it is important to elucidate the role of eIF2A in translation initiation. We aim to understand the detailed mechanism of eIF2A-mediated translation initiation by biochemical and structural approaches.

In this study, I have characterized the yeast eIF2A protein in-silico, biochemically and structurally. The computational analysis of eIF2A sequences from various organisms revealed unique features in eIF2A, namely nine-bladed beta-propeller domain, a conserved motif, stretch of positive residues, and C-terminal helices. It also revealed their diversity and presence of different isoforms of eIF2A in various organisms. Biochemically, I purified recombinant yeast eIF2A protein and detected interaction with 40S ribosomal subunit and initiator tRNA. Mutational studies were done to figure out the 40S and tRNA binding regions in eIF2A. eIF2A uses its unstructured middle regions and C-terminal helices for binding tRNA and 40S, respectively. The exact role of conserved beta-propellor of eIF2A remains to be deciphered.

Further, reconstitution of eIF2A complexes with 40S was used for structural studies by cryoEM. Multiple maps were reconstructed for different ribosomal complexes with eIF2A. These maps contain extra density, tentatively assigned to eIF2A, at the subunit interface of the head region of 40S. The ribosomal proteins present in the 40S head indeed bind with eIF2A in pull-down assays providing support to the assignment of extra density to eIF2A. This position of eIF2A is near the P site adjacent to tRNA and explains how eIF2A may deliver tRNA. Further, this position would have steric clash with the alpha subunit of the canonical eIF2 complex but not with any other eIFs. The insights helped us propose the mode of tRNA-delivery by eIF2A in translation initiation.