The following are the supplementary data related to this

The following are the supplementary data related to this article.
Conflict of interest
Acknowledgements This work was supported by JSPS KAKENHI Grant Numbers 24770068 and 15K07152.
Introduction Aminoacyl-tRNA synthetases (aaRSs) help maintain the genetic code by recognizing their cognate tRNAs and amino acids from a pool of competing reactants within the cell.1, 2 In the majority of cases, formation of aminoacylated tRNAs (charged tRNA) within the active site occurs via a two-step process (see Fig. 1). In the first step, amino Forskolin is activated by ATP, forming an aminoacyl adenylate and pyrophosphate. In the second step, the amino acid moiety on the adenylate is transferred to the 2′-hydroxyl group at the 3′ end of the tRNA, with simultaneous formation of an AMP product. We refer to the aaRS·tRNA complexes before and after amino acid transfer as the pre-transfer state and post-transfer state, respectively. Previous biochemical studies and crystal structures have provided valuable information about the first step of aminoacylation, the binding site of the adenylate, and the mode of interactions between identity elements on the tRNA and their binding partners on the aaRS (see Ibba et al. and references therein). More elusive are the details of how the charged tRNA dissociates from the aaRS prior to binding elongation factor Tu (EF-Tu) for delivery to the ribosome. In class I aaRSs, tRNA dissociation is the rate-determining step for tRNA aminoacylation, which has been shown to be stimulated in the presence of EF-Tu.4, 5 Dissociation has been hypothesized to begin with the charged 3′ end of the tRNA exiting the active site while the anticodon remains strongly bound to the aaRS. In this study, we investigate the series of events occurring in the active site that control tRNA dissociation. We use the structure of glutamyl-tRNA synthetase (GluRS) complexed with tRNAGlu from Thermus thermophilus as a representative of monomeric class I aaRSs. Although GluRS is atypical of class I aaRSs in that it requires tRNA to be bound before the aminoacyl adenylate can be formed, the final process of AMP and aminoacyl-tRNA dissociation involves an analogous set of molecules in all class I aaRSs. The modeled post-transfer states are differentiated by protonation of AMP and its neighboring amino acid residues and by the presence or the absence of AMP in the active site. These states have been selected based on suggested reaction mechanisms and internal pKa calculations. Through comparative analyses of each system state's behavior with the pre-transfer state and experimental results, undocking of AMP and changes in protonation states are evaluated as possible exit strategies for tRNA dissociation. Our results indicate that both factors assist in the release of the charged tRNA from the enzyme.
Results
Conclusion Experiments reveal that the complete dissociation of the charged tRNA from class I aaRSs takes place in the millisecond-to-second timescale and is stimulated by the presence of EF-Tu,4, 5 but our calculations indicate that there can be initial signs of tRNA release even at timescales of tens of nanoseconds. An important factor affecting the release of charged tRNA is the protonation state of residues in the active site of the aaRS. Results from network analysis, local nonbonded interaction energies, and free energies of binding all show that the Pre-transfer and Post(H-AMP) states form GluRS·tRNA interactions stronger than those of all other post-transfer states, regardless of the presence or the absence of AMP. The pKa calculations suggest that one of the α-ammonium hydrogens on the charging glutamate can transfer to the Glu41 side-chain carboxylate while in the active site. Glu41 is predicted to be a nearly universal handle that acts as a general base to facilitate tRNA release from the active site upon transfer of the amino acid. If Glu41 becomes solvated, either through removal of AMP or dissociation of the CCA hairpin, it would return to its charged state. Similarly, the pKa values measured at the beginning and at the end of the Post(No AMP/GluNH2) simulation shows that once the CCA end leaves the active site, breaking contacts with Glu41, the α-amino group on the charging glutamate can become reprotonated. From binding free energies, it appears that the pathways for AMP and tRNA dissociation are thermodynamically competitive, but once AMP has left the active site and the α-ammonium group is deprotonated, dissociation of the CCA hairpin can occur on the nanosecond timescale. Binding of EF-Tu to GluRS·tRNAGlu can stimulate tRNA release. Further studies will be needed to determine the molecular details of the migration of the tRNA to EF-Tu and whether its complete dissociation from the aaRS occurs prior to or during delivery of the charged tRNA to the ribosome A-site.