Cryo-EM study of an archaeal 30S initiation complex gives insights into evolution of translation initiation

Abstract

Archaeal translation initiation occurs within a macromolecular complex containing the small ribosomal subunit (30S) bound to mRNA, initiation factors aIF1, aIF1A and the ternary complex aIF2:GDPNP:Met-tRNA i Met. Here, we determine the cryo-EM structure of a 30S: mRNA:aIF1A:aIF2:GTP:Met-tRNA i Met complex from Pyrococcus abyssi at 3.2 Å resolution. It highlights archaeal features in ribosomal proteins and rRNA modifications. We find an aS21 protein, at the location of eS21 in eukaryotic ribosomes. Moreover, we identify an N-terminal extension of archaeal eL41 contacting the P site. We characterize 34 N 4-acetylcytidines distributed throughout 16S rRNA, likely contributing to hyperthermostability. Without aIF1, the 30S head is stabilized and initiator tRNA is tightly bound to the P site. A network of interactions involving tRNA, mRNA, rRNA modified nucleotides and C-terminal tails of uS9, uS13 and uS19 is observed. Universal features and domain-specific idiosyncrasies of translation initiation are discussed in light of ribosomal structures from representatives of each domain of life.

Cryo‐EM structure of the ribosome functional complex of the human pathogen Staphylococcus aureus at 3.2 Å resolution

“…However, several studies have also shown that loss of some particular rRNA modifications leads to diverse negative effects: severe growth retardation [30], slower rates and accuracy of translation [31], alterations of active sites [32], and a decrease in the fidelity of translation initiation [33]. Due to recent advances in X‐ray crystallography [2,8] and single‐particle electron [34,35] cryo‐EM, structural biology techniques have become efficient methods for verification rRNA modifications. Exceptional scientific rigor should be taken to cross‐validate with biochemical and bioinformatics data if available to ensure that errors do not occur.…”

Recent Advances in Archaeal Translation Initiation

“…No r-proteins found only in the archaeal and bacterial domains are found. One protein that could be specific of the archaeal domain found in place of eukaryotic eS21 and, therefore, named aS21 was identified recently in the SSU of Pyrococcus abyssi (Coureux et al, 2020) and Thermococcus celer (Nurenberg-Goloub et al, 2020). However, further phylogenetic studies are required to firmly determine whether the protein is unique to archaea or distantly related to eS21.…”

“…The number of universal (u), eukaryotic (e), bacterial (b), archaeal (a) -type ribosomal protein is indicated. One protein possibly specific to archaea (a) has recently been identified in P. abyssi (Coureux et al, 2020;Nurenberg-Goloub et al, 2020).…”

“…The cryo-EM structure of a TI complex from P. abyssi (Pa) using an mRNA derived from that of the gene coding for Pa-aEF1A containing a strong SD sequence [A(−17) UUUGGAGGUGAUUUAAA(+1)UGCCAAAG(+9)] is known at 3.4 Å resolution (Coureux et al, 2020). In the mRNA exit chamber, the SD duplex is extended to nine nucleotides and involves the 5′ AUCACCUCC 3′ sequence of the 3′-end of the 16S rRNA.…”

“…In bacteria, eS28 is absent and uS2 possesses a supplementary inserted helical domain occupying the position of eS17. Interestingly, comparison of the bacterial structures with the archaeal one showed that the spacing between the AUG codon and the SD sequence changed the position of the duplex in the chamber, probably explaining how it influences translation initiation efficiency (Coureux et al, 2020). Overall, archaeal and bacterial exit channels appear as two structural solutions for binding the SD duplex.…”