Aminoacyl-tRNA synthetases catalyze the attachment of specific amino acids to cognate tRNAs in a two-step process that is critical for the faithful translation of genetic information. During the first chemical step of tRNA aminoacylation, noncognate amino acids that are smaller than or isosteric with the cognate substrate can be misactivated. Thus, to maintain high accuracy during protein translation, some synthetases have evolved an editing mechanism. Previously, we showed that class II Escherichia coli proline-tRNA synthetase (ProRS) is capable of (1) weakly misactivating Ala, (2) hydrolyzing the misactivated Ala-AMP in a reaction known as pretransfer editing, and (3) deacylating a mischarged Ala-tRNA(Pro) variant via a post-transfer editing pathway. In contrast to most systems where an editing function has been established, pretransfer editing by E. coli ProRS occurs in a tRNA-independent fashion. However, neither the pre- nor the post-transfer editing active site(s) has been identified. Sequence analyses revealed that most prokaryotic ProRSs possess a large insertion domain (INS) between class II conserved motifs 2 and 3. The function of the approximately 180-amino acid INS in E. coli ProRS is the subject of this investigation. Alignment-guided Ala scanning mutagenesis was carried out to test conserved amino acid residues present in the INS for their role in pre- and post-transfer editing. Our biochemical data and modeling studies suggest that the prokaryotic INS plays a critical role in editing and that this activity resides in a domain that is functionally and structurally distinct from the aminoacylation active site.

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 posttransfer 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.

Molecular dynamics simulations of RNA microhelix Ala indicate that G:U and other 3:70 purine: pyrimidine wobble pairs induce local deviations from A-form geometry in their respective microhelices; the helix is underwound at the base-pair step above and overwound at the base-pair step below, in each case by about 7-9°compared to canonical A-form RNA. On the basis of analysis of average water densities and residence lifetimes, the wild-type microhelix strongly binds a water molecule in the minor groove of the 3:70 base pair, consistent with crystallographic analyses of an RNA duplex derived from the acceptor stem of Escherichia coli tRNA Ala . Other wobble pairs show water binding at this position but to a lesser degree; the strength of water binding correlates directly with the measured aminoacylation activities of the microhelices as substrates for E. coli alanyl-tRNA synthetase (G:U > 2AA:IsoC > G:dU > I:U). Watson-Crick base pairs at the 3:70 position show no tendency toward specific hydration. This tightly bound minor-groove water in the microhelices with 3:70 wobble pairs evidently does not function to stabilize a particular local helical structure, but it may play a role as a specific recognition element or serve as an indicator of interaction specificity between the microhelix and a hydrogen-bonding residue of the aminoacyl-tRNA synthetase.

Accuracy in translation of the genetic code into proteins depends upon correct tRNA–mRNA recognition in the context of the ribosome. In human tRNAUUULys,3 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 (ψ) at position 39—two of which, ms2t6A37 and mcm5s2U34, are required to achieve wild-type binding activity of wild-type human tRNAUUULys,3 [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 ψ39 most of the simulation time but weakly, as most of the residence lifetimes are <40 ps.