The CAR–mRNA Interaction Surface Is a Zipper Extension of the Ribosome A Site

Abstract

The ribosome CAR interaction surface behaves as an extension of the decoding center A site and has H-bond interactions with the +1 codon, which is next in line to enter the A site. Through molecular dynamic simulations, we investigated the codon sequence specificity of this CAR–mRNA interaction and discovered a strong preference for GCN codons, suggesting that there may be a sequence-dependent layer of translational regulation dependent on the CAR interaction surface. Dissection of the CAR–mRNA interaction through nucleotide substitution experiments showed that the first nucleotide of the +1 codon dominates over the second nucleotide position, consistent with an energetically favorable zipper-like activity that emanates from the A site through the CAR–mRNA interface. Moreover, the CAR/+1 codon interaction is affected by the identity of nucleotide 3 of +1 GCN codons, which influences the stacking of G and C. Clustering analysis suggests that the A-site decoding center adopts different neighborhood substates that depend on the identity of the +1 codon.

GNN codon adjacency regulates protein translation

“…MD was performed as described in Dalgarno et al . [7]. Briefly, we used a 494-residue subsystem of the ribosome consisting of the decoding center neighborhood.…”

“…The mechanisms by which pre-accommodation ribosomes (21-nt RFPs) are slowed by +1 GNN codons are unknown. However, once ribosomes have accommodated the correct A-site tRNA (28-nt RFPs), the effects of +1 GNN codons on translation rates are likely mediated by the ribosome CAR surface which is located next to the A-site tRNA anticodon during translocation [1,[7][8][9]. Supporting this hypothesis, MD analysis of the translocation ribosome decoding center with A-site NNU codons followed by +1 GCU or +1 CGU revealed significantly higher H-bond interactions between CAR and +1 GCU compared to +1 CGU (Fig 4A , B; Student's t-test p < 0.01).…”

“…This stacking of nt 34 with C1054 of CAR positions CAR as an extension of the anticodon poised to interact with the mRNA +1 codon. Molecular dynamics (MD) simulations of a subsystem of the decoding center neighborhood of translocating ribosomes revealed that the interaction between CAR and the +1 codon is particularly strong during stage II of translocation [9] and the interaction showed a remarkable sequence preference for GNN codons [7-9]. The discovery of CAR and its sequence-specificity for +1 GNN codons further supported the hypothesis that GNN codons might participate in a layer of protein translation regulation.…”

“…With publication of cryo-EM structures of translocating yeast ribosomes, Abeyrathne et al [6] noted hydrogen-bonding between 18S rRNA C1274 and the mRNA nucleotide following (3' of) the A-site codon. With further examination, we discovered that C1274 is part of CAR [1,[7][8][9], a three-residue surface that interacts with the full (+1) codon 3' adjacent to the A-site codon. CAR consists of yeast 18S rRNA C1274 and A1427-which are conserved in prokaryotes and eukaryotes and henceforth referred to as C1054 and A1196, the corresponding 16S rRNA residues in E. coli-and R146 of yeast ribosomal protein Rps3 which is only conserved in eukaryotes.…”

GNN Codon Adjacency Tunes Protein Translation

“…With publication of cryo-EM structures of translocating yeast ribosomes, Abeyrathne et al [ 6 ] noted hydrogen bonding between 18S rRNA C1274 and the mRNA nucleotide following (3′ of) the A-site codon. With further examination, we discovered that C1274 is part of CAR [ 1 , 7 , 8 , 9 ], a three-residue surface that interacts with the full (+1) codon 3′ adjacent to the A-site codon. CAR consists of yeast 18S rRNA C1274 and A1427—which are conserved in prokaryotes and eukaryotes and are henceforth referred to as C1054 and A1196, the corresponding 16S rRNA residues in E. coli —and R146 of the yeast ribosomal protein Rps3, which is only conserved in eukaryotes.…”