Anticodon nucleotide modifications affect translational tuning by the ribosomal CAR surface

Figures

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  Figure 1:. Modification of tRNA nucleotide 34. (A) Nucleotide 34 modifications and corresponding codons and anticodons analyzed in this MD study. (B) CAR is an extension of the A-site anticodon (tRNA nucleotides 34–36). (C, D) Unmodified (C) and galQ modified (D) nucleotide 34 are highlighted with Van der Waals spheres. (E) H-bonding between nucleotide 34 and the wobble nucleotide is shown in a time course of average H-bond counts per frame of MD trajectories with nucleotide 34 modifications. (F) The variances of the H-bond count distributions of MD replicate trajectories for unmodified (unmod) and modified (mod) nucleotide 34 show no significant differences.

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  Figure 2:. Modifications alter nucleotide 34 base pairing. H-bonding between nucleotide 34 and its base-pairing partner A-site codon nucleotide 3 was averaged across all the frames of 30 MD replicates for each structure. The x-axis (this figure and Figs. 3, 4 and 5) shows the first two nucleotides (N1N2) of the A-site codon which were tested with wobble and Watson-Crick base pairing of the third nucleotide (see Fig. 1A) and with +1 codons GCU or CGU. Bars corresponding to the modified structures are indicated (M; also for Figs. 3, 4 and 5). Student’s t-test comparisons between unmodified and modified nucleotide 34 H-bonding. p-values are denoted as: * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, ns p≥0.05 (no * sign).

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  Figure 3:. Modifications affect A-site H-bonding. (A) H-bonding by the A-site codon was calculated by summing each mRNA nucleotide’s H-bonding with its in-frame and (occasional) −1 and +1 frame partners (e.g. wobble nt H-bonding with tRNA nt35+nt34+C of CAR). Average values across 30 MD replicates were plotted for each structure. (B) The A site’s CAR sensitivity calculated by subtracting the average A-site H-bonding of all 30 +1 CGU replicate simulations from the A-site H-bond count for each individual +1 GCU MD replicate giving a distribution of 30 GCU-CGUavg difference values. The means and standard errors of these distributions are plotted. Student’s t-test p-values are denoted as: * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, ns p≥0.05 (no * sign).

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  Figure 4:. Modifications affect pi-pi stacking. Base stacking was assessed by measuring the distances between the centers of geometry (COG) of base rings3. (A) COG distance between nucleotide 35 and 34 of the tRNA anticodon. (B) COG distance between nucleotide 34 and the cytosine (C) of the CAR interaction surface. Student’s t-test p-values are denoted as: * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, ns p≥0.05 (no * sign). Black brackets compare modified and unmodified nucleotide 34 structures; red brackets compare +1 GCU with +1 CGU structures,

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  Figure 5:. Modifications affect CAR site H-bonding. (A) Average H-bonding levels between the CAR residues and the mRNA +1 codon sequences GCU and CGU for different A-site and nucleotide 34 modification contexts. (B) CAR site H-bonds (GCU-CGUavg) shows the CAR functionality range (see text). Student’s t-test p-values are denoted as: * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, ns p≥0.05 (no * sign).

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  Figure 6:. CAR mediated translational tuning and stress. (A) Dicodon analysis of published ribosome profiling data shows ribosomal densities (RD) at A-site UUU and UUC codons adjacent to +1 GNN or +1 (notG)NN codons. Two replicate experiments with unstressed yeast cells (wo1, wo2) were compared with cells exposed to oxidative stress (oxi1, oxi2) (upper graph). Assessing log2(RD w +1 GNN/RD wo +1 GNN) (lower graph) showed that UUU codons followed by +1 GNN exhibited higher ribosome densities, indicating slower translation, an effect that was enhanced in stressed cells. (B) Open reading frames (ORFs) in several species were analyzed to calculate the fractions of TTT codons that are followed by GNN codons for all genes (blue) and ribosomal protein genes (yellow). (C) Hypothetical model of sequence specific translational regulation under stress. Stress (blue arrow) dynamically regulates the levels of nucleotide 34 modifications which influence (red arrows) the functionality of A site, CAR site and their interplay (green arrows). The magnitudes of sequence specific CAR braking, as well as CAR mediated tuning of the A-site codon recognition interactions, alter under stress through the structural…