Characterization of the Cys-tRNA(Pro) editing mechanism and functional interactions of bacterial YbaK protein

Fidelity of translation is critical for viable cellular functions. Aminoacyl-tRNA synthetases (aaRSs) catalyze the formation of aminoacyl-tRNAs (aa-tRNAs), which are essential components for protein synthesis on the ribosome. To achieve high accuracy in aminoacylation, some synthetases have evolved proofreading functions, and in a few cases, these functions are carried out by freestanding proteins. Defects in the editing ability of aaRSs can cause mis-acylation of tRNAs, resulting in growth defects in Escherichia coli (E. coli), an apoptotic response, or neurodegeneration in mammalian cells. We are interested in the role of the editing domain of prolyl-tRNA synthetase and the YbaK protein in aa-tRNA channeling and quality control in translation. E. coli ProRS misactivates alanine and hydrolyzes mischarged Ala-tRNAPro using an editing active site (INS) that is distinct from the amino acid activation site. The enzyme is also unable to discriminate cysteine, which has a molecular volume similar to that of cognate proline, and efficiently synthesizes misacylated Cys-tRNAPro . Interestingly, while the ProRS editing domain does not clear this mischarged product, the small freestanding YbaK protein, which is a homolog of the INS domain, deacylates Cys-tRNAPro.;In this work, we investigated the mechanism of deacylation and substrate specificity of YbaK at the molecular level, and the functional interactions of a ProRS˙YbaK complex both in vitro and in vivo. To understand the chemical basis of the distinct substrate specificities of these homologous editing domains, we investigated the mechanism of YbaK hydrolysis. Site-directed mutagenesis was carried out at conserved residues in the YbaK superfamily, as well as residues identified by computational docking studies and molecular dynamics simulations. Moreover, substrate specificity of YbaK was probed using isosteric amino acids. Our data support a mechanism of catalysis that exploits the unique chemistry of the substrate sidechain thiol. Taken together, these studies allow us to propose a novel "triple-sieve" mechanism of editing in which the INS domain of ProRS and YbaK co-evolved distinct mechanisms involving steric exclusion and thiol specific chemistry, respectively, to ensure accurate decoding of Pro codons.;YbaK is known as a general Cys-tRNA deacylase in vitro, deacylating both mischarged Cys-tRNAPro and correctly charged Cys-tRNACys. Earlier work suggested that the substrate specificity of YbaK editing is ensured through the formation of a ProRS˙tRNA Pro˙YbaK ternary complex in vitro. To understand the functional interactions between ProRS and YbaK, competition assays in the presence of elongation factor-Tu (EF-Tu) were carried out. These studies suggest that YbaK compensates for a lack of tRNA recognition by interacting with the corresponding aaRS, either CysRS or ProRS. Split-GFP reassembly results support the interactions between ProRS, CysRS, and YbaK in the cell. Crystallization of the ProRS˙tRNAPro˙YbaK ternary complex was also initiated to understand the interactions of ProRS and YbaK at atomic resolution.