Cryo-EM study of start codon selection during archaeal translation initiation

Abstract

Eukaryotic and archaeal translation initiation complexes have a common structural core comprising e/aIF1, e/aIF1A, the ternary complex (TC, e/aIF2-GTP-Met-tRNAiMet) and mRNA bound to the small ribosomal subunit. e/aIF2 plays a crucial role in this process but how this factor controls start codon selection remains unclear. Here, we present cryo-EM structures of the full archaeal 30S initiation complex showing two conformational states of the TC. In the first state, the TC is bound to the ribosome in a relaxed conformation with the tRNA oriented out of the P site. In the second state, the tRNA is accommodated within the peptidyl (P) site and the TC becomes constrained. This constraint is compensated by codon/anticodon base pairing, whereas in the absence of a start codon, aIF2 contributes to swing out the tRNA. This spring force concept highlights a mechanism of codon/anticodon probing by the initiator tRNA directly assisted by aIF2.

Archaeal and eukaryal translation initiation factor 1 differ in their RNA interacting loops

“…Similar to eIF1, the regions of aIF1 involved in interaction with the rRNA as well as the region which remains in close proximity to tRNA are positively charged. The presence of positively charged residues in the corresponding region of aIF1 confirms that it would bind to 30S small ribosomal subunit in a manner similar to that of eIF1 . Binding of aIF1 to the P site of 30S small ribosomal subunit would inhibit the interaction between the large and small subunits of ribosome.…”

“…The fact that aIF1 is functionally analogous to eIF1 has been well established . A recently solved cryo‐EM structure of an archaeal 30S initiation complex with aIF1 bound in a manner similar to eIF1 in 40S initiation complex further accentuates the fact . In P. horikoshii OT3, an ORF PH1771.1, encoding aIF1, has been annotated to be a homologue of eIF1 (also known as SUI1 in yeast) protein.…”

“…Among others, archaea possess an open reading frame (ORF) encoding archaeal translation initiation factor 1 (aIF1). The protein aIF1 has been shown to share a similar topographical localization as of eIF1 by binding to a position adjacent to the P site of 30S small ribosomal subunit . Furthermore, similar to the functions established by eIF1, aIF1 also aids in the binding of aIF2 to the ribosome as well as retains the ability to discriminate against noncanonical start codons .…”

Molecular analysis of the ribosome recycling factor ABCE 1 bound to the 30S post‐splitting complex

“…The molecular model of the small ribosomal subunit was built using the 70S model of P.fu . [4V6U (Armache et al , ), 5JBH (Coureux et al , )]. After rigid‐body fitting of the 30S into the density, the sequence was manually changed to T .…”

The structure of an E. coli tRNAfMet A1–U72 variant shows an unusual conformation of the A1–U72 base pair

“…Therefore, further structural studies of the TC are required to reveal the conformation of the A 1 -U 72 base pair of the initiator tRNA bound to e/aIF2. Finally, recent Cryo-EM studies of archaeal translation initiation complexes (Coureux et al 2016) show transient deformation of the TC structure at some stages of the translation initiation process. Resolutions of the cryo-EM structures were not sufficient to observe the status of the A 1 -U 72 base pair.…”