Abstract
In archaeal translation initiation, a preinitiation complex (PIC) made up of aIF1, aIF1A, the ternary complex (TC, e/aIF2-GTP-Met-tRNAiMet) and mRNA bound to the small ribosomal subunit is responsible for start codon selection. Many archaeal mRNAs contain a Shine-Dalgarno (SD) sequence allowing the PIC to be prepositioned in the vicinity of the start codon. Nevertheless, cryo-EM studies have suggested local scanning to definitely establish base pairing of the start codon with the tRNA anticodon. Here, using fluorescence anisotropy, we show that aIF1 and mRNA have synergistic binding to the Pyrococcus abyssi 30S. Stability of 30S:mRNA:aIF1 strongly depends on the SD sequence. Further, toeprinting experiments show that aIF1-containing PICs display a dynamic conformation with the tRNA not firmly accommodated in the P site. AIF1-induced destabilization of the PIC is favorable for proofreading erroneous initiation complexes. After aIF1 departure, the stability of the PIC increases reflecting initiator tRNA fully base-paired to the start codon. Altogether, our data support the idea that some of the main events governing start codon selection in eukaryotes and archaea occur within a common structural and functional core. However, idiosyncratic features in loop 1 sequence involved in 30S:mRNA binding suggest adjustments of e/aIF1 functioning in the two domains.
Highlights
Eukaryotic translation initiation involves a 43S preinitiation complex (PIC) consisting of the initiator methionyltRNA:eIF2:GTP ternary complex (TC) bound to the small ribosomal subunit in the presence of initiation factors 1, 1A, 5 and 3
It promotes TC binding to the ribosome [5,6], is necessary for mRNA scanning and essential to the accuracy of start codon selection by preventing translation initiation at non-AUG codons or at AUG codons in a non-optimal nucleotidic context [7,8,9,10,11]. eIF1 function was proposed to be associated with two distinct conformations of the ribosome, an ‘open’ scanning competent conformation and a ‘closed’ state unable to scan mRNA, only reached when a correct AUG codon has been found [8,12]
Recent cryo-EM studies of eukaryotic translation initiation complexes have highlighted some of the structural changes that lead to a closed state of the PIC with the initiator tRNA base-paired to the start codon [16,17,18,19]
Summary
Eukaryotic translation initiation involves a 43S preinitiation complex (PIC) consisting of the initiator methionyltRNA:eIF2:GTP ternary complex (TC) bound to the small ribosomal subunit in the presence of initiation factors (eIFs) 1, 1A, 5 and 3. EIF1 function was proposed to be associated with two distinct conformations of the ribosome, an ‘open’ scanning competent conformation and a ‘closed’ state unable to scan mRNA, only reached when a correct AUG codon has been found [8,12] Consistent with this idea, it was shown that final tRNA accommodation within the P site after start codon recognition was triggered by eIF1 departure [13]. Recent cryo-EM studies of eukaryotic translation initiation complexes have highlighted some of the structural changes that lead to a closed state of the PIC with the initiator tRNA base-paired to the start codon [16,17,18,19] These structures have shown how accommodation of the initiator tRNA at the P site, from a POUT to a PIN position [20] led to a closure of the mRNA channel in the 40S subunit, destabilizing eIF1 and provoking its departure
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
