CP1 domain of leucyl-tRNA synthetase: dissecting its dual roles in amino acid editing and RNA splicing

The essential family of aminoacyl-tRNA synthetase (AARS) enzymes catalyzes the attachment of an amino acid to its cognate tRNA during ribosome-based translation of mRNA. Leucyl-tRNA synthetase (LeuRS) ensures fidelity in protein synthesis via proofreading or editing mechanisms. The editing that hydrolyzes noncognate amino acids mischarged onto tRNALeu is called post-transfer editing. The hydrolytic post-transfer editing active site is located in a discretely folded polypeptide insertion called connective polypeptide 1 (CP1) that is linked to the enzyme’s main body by two flexible β-strand linkers. Disruption of the CP1 domain-based editing function in LeuRS results in amino acid toxicities that compromise cell viability. A fluorescence-based in vivo assay was designed to quantify the effects of editing defects and hence assess the limits of mistranslation that can be borne by the cell. Sequence enabled reassembly of N and C-terminal fragments of the green fluorescence protein (GFP) were studied in vivo in the presence of editing defective LeuRS and noncognate amino acids. In the yeast cytoplasmic LeuRS (ycLeuRS), the conserved post-transfer editing pocket is the target binding site for a novel class of benzoxaborole-based antimicrobials that trap tRNALeu and halt protein synthesis. Resistance mutations (D487G and D487N) to the antimicrobial compound AN2690 lie outside the drug binding pocket and provided a unique opportunity to study editing mechanisms in the ycLeuRS. The Asp487 residue is located in a CP1 domain-based eukaryote-specific flexible insert called I4 that forms a ‘cap’ over the benzoxaborole-AMP adduct bound in the CP1 domain editing active site. Mutational and biochemical analysis at Asp487 identified a salt bridge between Asp487 and Arg316 in the hinge region of the I4 cap that is critical to tRNA deacylation. Thus, this electrostatic interaction stabilizes the cap during binding of the editing substrate for hydrolysis in the ycLeuRS. An alternative pre-transfer editing pathway has also been identified in LeuRS and cleaves the noncognate amino acid before it is transferred to tRNALeu, at the stage of aminoacyl-AMP. Co-existence of both pre- and post-transfer editing pathways was highlighted in the ycLeuRS, as has also been shown earlier for E. coli LeuRS. Detailed biochemical investigations on the editing activity of this enzyme revealed that ycLeuRS shifts between the two editing pathways and this shift is dictated by the chemical identity of the noncognate amino acid misactivated by the enzyme. While isoleucine is mainly cleared via the post-transfer editing route that targets Ile-tRNALeu, methionine is edited via the pre-transfer pathway by hydrolysis of methionyl-adenylate in ycLeuRS. The yeast mitochondrial LeuRS (ymLeuRS) was recruited to perform an alternate cellular role of mRNA splicing. Splicing-sensitive sites have been located within and and in close proximity to the CP1 domain. Remarkably, E. coli LeuRS supports splicing in vivo, although its CP1 domain appears to lack the finer adaptations for efficient splicing compared to its counterpart from the ymLeuRS. In vitro and in vivo analysis dissected functional divergences of the ymLeuRS CP1 domain that accommodate an alternate cellular role for RNA splicing at the expense of its housekeeping aminoacylation and editing function. A close look at the connecting β-strands between the CP1 domain and main body highlighted that these β-strands, as well short extensions into the enzyme main body, are indispensable to not only LeuRS’s editing function, but also to its splicing activity