Influence of Sequence and Covalent Modifications on Yeast tRNA Dynamics

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.

修饰核苷酸在转运RNA（transfer RNA, tRNA）中广泛存在。已有实验研究表明，这些共价修饰在调控转运RNA功能方面发挥着重要作用。本研究采用分子动力学（molecular dynamics, MD）模拟方法，探究修饰如何改变转运RNA的动态特性。本研究以带有修饰与未带有修饰的天冬氨酸转运RNA（tRNA-Asp）、苯丙氨酸转运RNA（tRNA-Phe）以及起始甲硫氨酸转运RNA（tRNA-iMet）的X射线晶体结构，作为采用AMBER软件进行333纳秒（ns）显式溶剂分子动力学模拟的初始结构。针对每种转运RNA分子，均开展三次独立的轨迹计算，最终六个分子的总分子动力学模拟时长累计达6微秒（μs）。原子位置的整体均方根偏差（root-mean-square deviation, RMSD）分析结果显示，修饰仅对苯丙氨酸转运RNA（tRNA-Phe）的整体结构带来显著的刚性化效应。有趣的是，反密码子茎环（anticodon stem-loop, ASL）的均方根偏差结果表明，在该结构域中，带有修饰的转运RNA比未修饰的转运RNA结构更为刚性。但值得注意的是，带有修饰的转运RNA其反密码子区域的均方根偏差，却高于对应未修饰的转运RNA。上述研究结果表明，反密码子茎环的刚性可通过修饰进行精细调控：反密码子臂的刚性对于转运RNA在核糖体中的移位至关重要，而反密码子的柔性则对密码子识别具有重要意义。研究还评估了带有修饰的转运RNA中假尿苷的糖环折叠方式与水停留时间，以及未修饰转运RNA中对应尿苷的相关参数，结果证实假尿苷更倾向于采用3′-内式构象，且与水分子相互作用的倾向更强。本研究采用主成分分析（principal component analysis, PCA），分析转运RNA中的协同运动模式。此外，本研究还对同一分子的不同轨迹，以及带有修饰与未修饰转运RNA的轨迹之间的主成分分析协方差重叠度进行了比较。该比较结果表明，修饰会改变转运RNA的协同运动模式。针对反密码子碱基，本研究比较了带有修饰与未修饰分子的碱基堆积程度，结果发现仅未修饰的天冬氨酸转运RNA（tRNA-Asp）具有显著更高的碱基堆积时间占比。总体而言，本研究的模拟结果表明，共价修饰对转运RNA动态特性的影响并非单一模式：修饰可使结构的某些区域柔性增强，同时使另一些区域刚性提升。