A Ribosome Interaction Surface Sensitive to mRNA GCN Periodicity

Abstract

A longstanding challenge is to understand how ribosomes parse mRNA open reading frames (ORFs). Significantly, GCN codons are over-represented in the initial codons of ORFs of prokaryote and eukaryote mRNAs. We describe a ribosome rRNA-protein surface that interacts with an mRNA GCN codon when next in line for the ribosome A-site. The interaction surface is comprised of the edges of two stacked rRNA bases: the Watson–Crick edge of 16S/18S rRNA C1054 and the adjacent Hoogsteen edge of A1196 (Escherichia coli 16S rRNA numbering). Also part of the interaction surface, the planar guanidinium group of a conserved Arginine (R146 of yeast ribosomal protein Rps3) is stacked adjacent to A1196. On its other side, the interaction surface is anchored to the ribosome A-site through base stacking of C1054 with the wobble anticodon base of the A-site tRNA. Using molecular dynamics simulations of a 495-residue subsystem of translocating ribosomes, we observed base pairing of C1054 to nucleotide G at position 1 of the next-in-line codon, consistent with previous cryo-EM observations, and hydrogen bonding of A1196 and R146 to C at position 2. Hydrogen bonding to both of these codon positions is significantly weakened when C at position 2 is changed to G, A or U. These sequence-sensitive mRNA-ribosome interactions at the C1054-A1196-R146 (CAR) surface potentially contribute to the GCN-mediated regulation of protein translation.

Arginine Methylation Regulates Ribosome CAR Function

“…A key aspect of CAR function is that the interaction between CAR and the +1 codon is sensitive to the sequence at the +1 codon. Using N1, we showed previously that GCU at the +1 codon leads to strong CAR/+1 codon interactions, whereas replacement of the second nucleotide (GGU, GAU or GUU) led to significantly reduced interactions [12]. The same relationship was observed with N2 ( Figure S5) confirming our previous conclusion that the CAR interaction is sequence sensitive with a preference for GCN codons, and suggesting that sensitivity to translation regulation through the CAR interface likely depends on the degree to which codons in a gene's ORF conform to GCN.…”

“…Modulation of these interactions provides potential pathways for fine-tuning rates of protein translation in different cellular conditions. Cryo-EM studies in yeast have identified five intermediate stages of ribosome translocation (stages I through V; [17]) and we have identified a ribosome interaction surface, named CAR [12,18], that has pronounced hydrogen-bonding to the mRNA during the early translocation stages I and II. CAR interacts with the +1 codon next in line to enter the ribosome A site.…”

“…This has included the use of ribosome subsystems to characterize important steps in recognition of the correct tRNA through interactions of 16S/18S A1492, A1493 and G530 (E. coli numbering) with the minor groove of the codon-anticodon base pairs in the A-site decoding center [22,24,25]. In a previous study [12], we characterized interactions of the CAR surface with the +1 codon next to enter the A site. We used a subsystem of the yeast ribosome with 495 residues-183 rRNA nucleotides and 312 ribosomal protein amino acids-and applied a restraining force on an "onion shell" of residues around the surface of the subsystem in order to maintain the topologies and structures of the different translocation stages of the yeast ribosome identified in cryo-EM studies [12].…”

“…R146 is conserved across eukaryotes, but not prokaryotes [12,18]. Mutational analysis has shown that when R146 is replaced in yeast Rps3, this can lead to lethal or slow growth phenotypes depending on the amino acid substitution, and R146 substitution can affect translation initiation and fidelity [38,39].…”

“…Methylation of rRNA or mRNA nucleotides can also modulate translation initiation or elongation [9][10][11]. In a recent study [12], we identified the CAR ribosome surface, which has sequence-sensitive interactions with the mRNA as it enters the ribosome decoding center during translation elongation. In this study, we elucidate a mechanism to modulate CAR function based on cellular conditions.…”