Characterization of leucyl-tRNA synthetase from Escherichia coli and Saccharomyces cerevisiae in aminoacylation, amino acid editing, and protein-dependent RNA splicing

Aminoacyl-tRNA synthetases (aaRS) are an essential family of enzymes responsible for aminoacylation of tRNA during protein synthesis. Remarkably, leucyl-tRNA synthetase (LeuRS) is not only important for protein synthesis but has also adapted for a secondary role in RNA splicing. Recently, the x-ray co-crystal structure of T. thermophilus LeuRS revealed a unique C-terminal extension of about sixty amino acids that forms a discrete domain and interacts with the corner of the L-shaped tRNALeu. It is hypothesized that the C-terminal domain may potentially be important to RNA-protein interactions in tRNA recognition as well as RNA splicing.;Deletion of the C-terminal domain abolished both aminoacylation and amino acid editing activities of the Escherichia coli LeuRS, but surprisingly enhanced for the yeast mitochondrial enzyme. Extensive mutagenesis and characterization also suggested that the overall contour of the LeuRS C-terminal domain rather than specific amino acid residues was important for RNA-protein interactions. Interestingly, regions near the C-terminus of yeast mitochondrial LeuRS have also been implicated in RNA splicing.;Chimeric LeuRSs demonstrated that the E. coli and yeast C-terminal domains were interchangeable and conferred wild-type-like enzymatic activities. Secondary structure analysis by circular dichroism showed that the overall alpha-helical character of the yeast enzyme was increased either in the absence of its C-terminal domain or in the presence of KCl. In addition, the C-terminal linker which tethered the C-terminal domain to the main body was also important to aminoacylation activity of LeuRS.;Unlike E. coli tRNALeu, deletion of the anticodon stem-loop of ymtRNALeu nearly abolished aminoacylation activity compared to the wild-type tRNA. However, deletion of the variable stem-loop did not significantly affect aminoacylation activity. Surprisingly, similar experiments using the yeast C-terminal deletion mutant severely impaired aminoacylation activity in all tRNA deletion mutants.;Using extensive deletion and site-directed mutagenesis, we characterized the C-terminal domain of LeuRSs from different origins to understand its role. The results demonstrated that the native environment of the enzyme influenced the functional role of the unique C-terminal domain of both LeuRSs. These combined biochemical investigations suggest that the yeast mitochondrial LeuRS C-terminal domain has uniquely adapted to accommodate aminoacylation, amino acid editing, and RNA-protein interactions.