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The role of ribonucleic acid (RNA) in biology continues to grow, but insight into important aspects of RNA behavior is lacking, such as dynamic structural ensembles in different environments, how flexibility is coupled to function, and how function might be modulated by small molecule binding. In the case of proteins, much progress in these areas has been made by complementing experiments with atomistic simulations, but RNA simulation methods and force fields are less mature. It remains challenging to generate stable RNA simulations, even for small systems where well-defined, thermostable structures have been established by experiments. Many different aspects of RNA energetics have been adjusted in force fields, seeking improvements that are transferable across a variety of RNA structural motifs. In this work, the role of weak CH···O interactions is explored, which are ubiquitous in RNA structure but have received less attention in RNA force field development. By comparing data extracted from high-resolution RNA crystal structures to energy profiles from quantum mechanics and force field calculations, it is shown that CH···O interactions are overly repulsive in the widely used Amber RNA force fields. A simple, targeted adjustment of CH···O repulsion that leaves the remainder of the force field unchanged was developed. Then, the standard and modified force fields were tested using molecular dynamics (MD) simulations with explicit water and salt, amassing over 300 μs of data for multiple RNA systems containing important features such as the presence of loops, base stacking interactions as well as canonical and noncanonical base pairing. In this work and others, standard force fields lead to reproducible unfolding of the NMR-based structures. Including a targeted CH···O adjustment in an otherwise identical protocol dramatically improves the outcome, leading to stable simulations for all RNA systems tested.

Transfer RNA (tRNA) anticodons adopt a highly ordered 3'-stack without significant base overlap. Density functional theory at the M06-2X/6-31+G(d,p) level in combination with natural bond orbital analysis was utilized to calculate the intramolecular interactions within the tRNA anticodon that are responsible for stabilizing the stair-stepped conformation. Ten tRNA X-ray crystal structures were obtained from the PDB databank and were trimmed to include only the anticodon bases. Hydrogenic positions were added and optimized for the structures in the stair-stepped conformation. The sugar-phosphate backbone has been retained for these calculations, revealing the role it plays in RNA structural stability. It was found that electrostatic interactions between the sugar-phosphate backbone and the base provide the most stability, rather than the traditionally studied interbase stacking. Base-stacking interactions, though present, were weak and inconsistent. Aqueous solvation was found to have little effect on the intramolecular interactions.

Aminoacyl-tRNA synthetases catalyze the attachment of cognate amino acids to specific tRNA molecules. To prevent potential errors in protein synthesis caused by misactivation of noncognate amino acids, some synthetases have evolved editing mechanisms to hydrolyze misactivated amino acids (pre-transfer editing) or misacylated tRNAs (post-transfer editing). In the case of post-transfer editing, synthetases employ a separate editing domain that is distinct from the site of amino acid activation, and the mechanism is believed to involve shuttling of the flexible CCA-3' end of the tRNA from the synthetic active site to the site of hydrolysis. The mechanism of pre-transfer editing is less well understood, and in most cases, the exact site of pre-transfer editing has not been conclusively identified. Here, we probe the pre-transfer editing activity of class II prolyl-tRNA synthetases from five species representing all three kingdoms of life. To locate the site of pre-transfer editing, truncation mutants were constructed by deleting the insertion domain characteristic of bacterial prolyl-tRNA synthetase species, which is the site of post-transfer editing, or the N- or C-terminal extension domains of eukaryotic and archaeal enzymes. In addition, the pre-transfer editing mechanism of Escherichia coli prolyl-tRNA synthetase was probed in detail. These studies show that a separate editing domain is not required for pre-transfer editing by prolyl-tRNA synthetase. The aminoacylation active site plays a significant role in preserving the fidelity of translation by acting as a filter that selectively releases non-cognate adenylates into solution, while protecting the cognate adenylate from hydrolysis.

Accuracy in translation of the genetic code into proteins depends upon correct tRNA-mRNA recognition in the context of the ribosome. In human tRNA(Lys,3)UUU three modified bases are present in the anticodon stem-loop--2-methylthio-N6-threonylcarbamoyladenosine at position 37 (ms2t6A37), 5-methoxycarbonylmethyl-2-thiouridine at position 34 (mcm5s2U34) and pseudouridine (psi) at position 39--two of which, ms2t6A37 and mcm5s2U34, are required to achieve wild-type binding activity of wild-type human tRNA(Lys,3)UUU [C. Yarian, M. Marszalek, E. Sochacka, A. Malkiewicz, R. Guenther, A. Miskiewicz and P. F. Agris (2000) Biochemistry, 39, 13390-13395]. Molecular dynamics simulations of nine tRNA anticodon stem-loops with different combinations of nonstandard bases were performed. The wild-type simulation exhibited a canonical anticodon stair-stepped conformation. The ms2t6 modification at position 37 is required for maintenance of this structure and reduces solvent accessibility of U36. Ms2t6A37 generally hydrogen bonds across the loop and may prevent U36 from rotating into solution. A water molecule does coordinate to psi39 most of the simulation time but weakly, as most of the residence lifetimes are <40 ps.

Specific aminoacylation by aminoacyl-tRNA synthetases requires accurate recognition of cognate tRNA substrates. In the case of alanyl-tRNA synthetase (AlaRS), RNA duplexes that mimic the acceptor stem of the tRNA are efficient substrates for aminoacylation in vitro. It was previously shown that recognition by AlaRS is severely affected by a simple base pair transversion of the G2:C71 pair at the second position in the RNA helix. In this study, we determined the aminoacylation efficiencies of 50 variants of the tRNA(Ala) acceptor stem containing substitutions at the 2:71 position. We find that there is not a single functional group of the wild-type G2:C71 base pair that is critical for positive recognition. Rather, we observed that base-pair orientation plays an important role in recognition. In particular, pyrimidine2:purine71 combinations generally resulted in decreased aminoacylation efficiency compared to the corresponding purine:pyrimidine pair. Moreover, the activity of a pyrimidine:purine variant could be partially restored by the presence of a major groove amino group at position 71. In an attempt to understand this result further, dielectric continuum electrostatic calculations were carried out, in some cases with additional inclusion of van der Waals interaction energies, to determine interaction potentials of the wild-type duplexAla and seven 2:71 variants. This analysis revealed a positive correlation between major groove negative electrostatic potential in the vicinity of the 3:70 base pair and measured aminoacylation efficiency.

Transfer of alanine from Escherichia coli alanyl-tRNA synthetase (AlaRS) to RNA minihelices that mimic the amino acid acceptor stem of tRNA(Ala) has been shown, by analysis of variant minihelix aminoacylation activities, to involve a transition state sensitive to changes in the 'discriminator' base at position 73. Solution NMR has indicated that this single-stranded nucleotide is predominantly stacked onto G1 of the first base pair of the alanine acceptor stem helix. We report the activity of a new variant with the adenine at position 73 substituted by its non-polar isostere 4-methylindole (M). Despite lacking N7, this analog is well tolerated by AlaRS. Molecular dynamics (MD) simulations show that the M substitution improves position 73 base stacking over G1, as measured by a stacking lifetime analysis. Additional MD simulations of wild-type microhelix(Ala) and six variants reveal a positive correlation between N73 base stacking propensity over G1 and aminoacylation activity. For the two DeltaN7 variants simulated we found that the propensity to stack over G1 was similar to the analogous variants that contain N7 and we conclude that the decrease in aminoacylation efficiency observed upon deletion of N7 is likely due to loss of a direct stabilizing interaction with the synthetase.