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. 2014 Aug 12;10(8):3473-3483.
doi: 10.1021/ct500107y. Epub 2014 May 28.

Influence of Sequence and Covalent Modifications on Yeast tRNA Dynamics

Xiaoju Zhang  1 , Ross C Walker  2 , Eric M Phizicky  1 , David H Mathews  1
Affiliations

Influence of Sequence and Covalent Modifications on Yeast tRNA Dynamics

Xiaoju Zhang et al. J Chem Theory Comput. .

Abstract

Modified nucleotides are prevalent in tRNA. Experimental studies reveal that these covalent modifications play an important role in tuning tRNA function. In this study, molecular dynamics (MD) simulations were used to investigate how modifications alter tRNA dynamics. The X-ray crystal structures of tRNA(Asp), tRNA(Phe), and tRNA(iMet), both with and without modifications, were used as initial structures for 333 ns explicit solvent MD simulations with AMBER. For each tRNA molecule, three independent trajectory calculations were performed, giving an aggregate of 6 μs of total MD across six molecules. The global root-mean-square deviations (RMSD) of atomic positions show that modifications only introduce significant rigidity to the global structure of tRNA(Phe). Interestingly, RMSDs of the anticodon stem-loop (ASL) suggest that modified tRNA has a more rigid structure compared to the unmodified tRNA in this domain. The anticodon RMSDs of the modified tRNAs, however, are higher than those of corresponding unmodified tRNAs. These findings suggest that the rigidity of the anticodon stem-loop is finely tuned by modifications, where rigidity in the anticodon arm is essential for tRNA translocation in the ribosome, and flexibility of the anticodon is important for codon recognition. Sugar pucker and water residence time of pseudouridines in modified tRNAs and corresponding uridines in unmodified tRNAs were assessed, and the results reinforce that pseudouridine favors the 3'-endo conformation and has a higher tendency to interact with water. Principal component analysis (PCA) was used to examine correlated motions in tRNA. Additionally, covariance overlaps of PCAs were compared for trajectories of the same molecule and between trajectories of modified and unmodified tRNAs. The comparison suggests that modifications alter the correlated motions. For the anticodon bases, the extent of stacking was compared between modified and unmodified molecules, and only unmodified tRNA(Asp) has significantly higher percentage of stacking time. Overall, the simulations reveal that the effect of covalent modification on tRNA dynamics is not simple, with modifications increasing flexibility in some regions of the structure and increasing rigidity in other regions.

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Figures

Figure 1
Figure 1
Backbone heavy atom RMSD as a function of time for tRNA molecules. The atoms included are P, O5′, C5′, C4′, C3′, and O3′. The left column shows unmodified tRNAs, and the right column shows corresponding modified tRNAs. From top to bottom, tRNAAsp, tRNAPhe, and tRNAiMet plots are shown, and each panel has three independent trajectories plotted in red, yellow, and blue. The average RMSDs of modified tRNAAsp and unmodified tRNAAsp are 5.45 ± 0.35 and 5.53 ± 0.17 Å. The average RMSDs of modified tRNAPhe and unmodified tRNAPhe are 3.67 ± 0.041 and 4.32 ± 0.095 Å. The average RMSDs of modified tRNAiMet and unmodified tRNAiMet are 4.61 ± 0.20 and 4.64 ± 0.25 Å.
Figure 2
Figure 2
Backbone heavy atom RMSD as a function of time for tRNA anticodon stem loops (nucleotides 27–43). The left column shows unmodified tRNAs, and the right column shows corresponding modified tRNAs. From top to bottom, tRNAAsp, tRNAPhe, and tRNAiMet plots are provided, and each panel has three independent trajectories plotted in red, yellow, and blue, where the trajectory colors correspond to those in Figure 1. The average RMSDs of modified tRNAAsp and unmodified tRNAAsp are 2.44 ± 0. 030 and 4.00 ± 0.32 Å. The average RMSDs of modified tRNAPhe and unmodified tRNAPhe are 2.07 ± 0.21 and 3.84 ± 0.12 Å. The average RMSDs of modified tRNAiMet and unmodified tRNAiMet are 3.70 ± 0.75 and 2.90 ± 0.23 Å.
Figure 3
Figure 3
All-atom RMSD as a function of time for tRNA anticodon loops. The left column shows unmodified tRNAs, and the right column shows corresponding modified tRNAs. From top to bottom, tRNAAsp, tRNAPhe, and tRNAiMet plots are listed, and each panel has three independent trajectories plotted in red, yellow and blue, where the trajectory colors correspond to those in Figure 1. The average RMSDs of modified tRNAAsp and unmodified tRNAAsp are 3.81 ± 0.68 and 3.09 ± 0.52 Å. The average RMSDs of modified tRNAPhe and unmodified tRNAPhe are 2.77 ± 0.24 and 2.57 ± 0.82 Å. The average RMSDs of modified tRNAiMet and unmodified tRNAiMet are 3.40 ± 0.59 and 2.67 ± 0.20 Å.
Figure 4
Figure 4
Averaged distribution of U or Ψ base water residence time for the water molecules in the simulation system. The distributions are averaged from three trajectories of each molecule, and ± a single standard error is displayed as error bars. To focus on longer residence time range, the time axes in the plots are truncated from 20 to 600 ps. Plots for the full time range (5 to 600 ps) are shown in Supporting Information Figure S2. Panel A: From top to bottom, plots for bases at position 13, 32, and 55 of tRNAAsp are listed, with unmodified tRNAAsp plotted in blue and modified tRNAAsp plotted in yellow. Panel B: From top to bottom, plots for bases at position 39 and 55 tRNAPhe are listed; unmodified tRNAPhe is plotted in blue, and modified tRNAPhe is plotted in yellow.
Figure 5
Figure 5
Fraction of stacking conformation during simulations for anticodon loop bases. Three stacks are shown for each molecule. From left to right are unmodified tRNAAsp, modified tRNAAsp, unmodified tRNAPhe, modified tRNAPhe, unmodified tRNAiMet, and modified tRNAiMet. In each panel, base 34 to 35 (blue), base 35 to 36 (orange), and base 36 to 37 (yellow) stacking conformation fractions are displayed.
Figure 6
Figure 6
Averaged covariance overlap between PCA of backbone heavy atoms. From left to right, three sets of bars represent tRNAAsp, tRNAPhe, and tRNAiMet, respectively. The blue bar is the averaged covariance between trajectories of unmodified molecule, the orange bar is the averaged covariance between trajectories of modified molecules, and the yellow bar is the averaged covariance between trajectories of unmodified molecule and modified molecule. Mean values are plotted, and a single standard deviation is shown as error bars.
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