Duplication of leucyl-tRNA synthetase in an archaeal extremophile may play a role in adaptation to variable environmental conditions.

Aminoacyl-tRNA synthetases (aaRSs) are ancient enzymes that play a fundamental role in protein synthesis. They catalyze the esterification of specific amino acids to the 3'-end of their cognate tRNAs and therefore play a pivotal role in protein synthesis. Although previous studies suggest that aaRS-dependent errors in protein synthesis can be beneficial to some microbial species, evidence that reduced aaRS fidelity can be adaptive is limited. Using bioinformatics analyses, we identified two distinct leucyl-tRNA synthetase (LeuRS) genes within all genomes of the archaeal family Sulfolobaceae. Remarkably, one copy, designated LeuRS-I, had key amino acid substitutions within its editing domain that would be expected to disrupt hydrolytic editing of mischarged tRNALeu and to result in variation within the proteome of these extremophiles. We found that another copy, LeuRS-F, contains canonical active sites for aminoacylation and editing. Biochemical and genetic analyses of the paralogs within Sulfolobus islandicus supported the hypothesis that LeuRS-F, but not LeuRS-I, functions as an essential tRNA synthetase that accurately charges leucine to tRNALeu for protein translation. Although LeuRS-I was not essential, its expression clearly supported optimal S. islandicus growth. We conclude that LeuRS-I may have evolved to confer a selective advantage under the extreme and fluctuating environmental conditions characteristic of the volcanic hot springs in which these archaeal extremophiles reside.

Coexistence of bacterial leucyl-tRNA synthetases with archaeal tRNA binding domains that distinguish tRNA(Leu) in the archaeal mode.

Leucyl-tRNA (transfer RNA) synthetase (LeuRS) is a multi-domain enzyme, which is divided into bacterial and archaeal/eukaryotic types. In general, one specific LeuRS, the domains of which are of the same type, exists in a single cell compartment. However, some species, such as the haloalkaliphile Natrialba magadii, encode two cytoplasmic LeuRSs, NmLeuRS1 and NmLeuRS2, which are the first examples of naturally occurring chimeric enzymes with different domains of bacterial and archaeal types. Furthermore, N. magadii encodes typical archaeal tRNA(Leu)s. The tRNA recognition mode, aminoacylation and translational quality control activities of these two LeuRSs are interesting questions to be addressed. Herein, active NmLeuRS1 and NmLeuRS2 were successfully purified after gene expression in Escherichia coli. Under the optimized aminoacylation conditions, we discovered that they distinguished cognate NmtRNA(Leu) in the archaeal mode, whereas the N-terminal region was of the bacterial type. However, NmLeuRS1 exhibited much higher aminoacylation and editing activity than NmLeuRS2, suggesting that NmLeuRS1 is more likely to generate Leu-tRNA(Leu) for protein biosynthesis. Moreover, using NmLeuRS1 as a model, we demonstrated misactivation of several non-cognate amino acids, and accuracy of protein synthesis was maintained mainly via post-transfer editing. This comprehensive study of the NmLeuRS/tRNA(Leu) system provides a detailed understanding of the coevolution of aminoacyl-tRNA synthetases and tRNA.

A Flexible peptide tether controls accessibility of a unique C-terminal RNA-binding domain in leucyl-tRNA synthetases.

A unique C-terminal domain extension is required by most leucyl-tRNA synthetases (LeuRS) for aminoacylation. In one exception, the enzymatic activity of yeast mitochondrial LeuRS is actually impeded by its own C-terminal domain. It was proposed that the yeast mitochondrial LeuRS has compromised its aminoacylation activity to some extent and adapted its C terminus for a second role in RNA splicing, which is also essential. X-ray crystal structures of the LeuRS-tRNA complex show that the 60 residue C-terminal domain is tethered to the main body of the enzyme via a flexible peptide linker and allows interactions with the tRNA(Leu) elbow. We hypothesized that this short peptide linker would facilitate rigid body movement of the C-terminal domain as LeuRS transitions between an aminoacylation and editing complex or, in the case of yeast mitochondrial LeuRS, an RNA splicing complex. The roles of the C-terminal linker peptide for Escherichia coli and yeast mitochondrial LeuRS were investigated via deletion mutagenesis as well as by introducing chimeric swaps. Deletions within the C-terminal linker of E. coli LeuRS determined that its length, rather than its sequence, was critical to aminoacylation and editing activities. Although deletions in the yeast mitochondrial LeuRS peptide linker destabilized the protein in general, more stable chimeric enzymes that contained an E. coli LeuRS C-terminal domain showed that shortening its tether stimulated aminoacylation activity. This suggested that limiting C-terminal domain accessibility to tRNA(Leu) facilitates its role in protein synthesis and may be a unique adaptation of yeast mitochondrial LeuRS that accommodates its second function in RNA splicing.

Expression and characterization of the human mitochondrial leucyl-tRNA synthetase.

A cDNA clone encoding the human mitochondrial leucyl-tRNA synthetase (mtLeuRS) has been identified from the EST databases. Analysis of the protein encoded by this cDNA indicates that the protein is 903 amino acids in length and contains a mitochondrial signal sequence that is predicted to encompass the first 21 amino acids. Sequence analysis shows that this protein contains the characteristic motifs of class I aminoacyl-tRNA synthetases and regions of high homology to other mitochondrial and bacterial LeuRS proteins. The mature form of this protein has been cloned and expressed in Escherichia coli. Gel filtration indicates that human mtLeuRS is active in a monomeric state, with an apparent molecular mass of 101 kDa. The human mtLeuRS is capable of aminoacylating E. coli tRNA(Leu). Its activity is inhibited at high levels of either monovalent or divalent cations. K(M) and k(cat) values for ATP:PP(i) exchange and for the aminoacylation reaction have been determined.

Leucyl-tRNA and lysyl-tRNA synthetases, derived from the high-Mr complex of sheep liver, are hydrophobic proteins.

The leucyl-tRNA and lysyl-tRNA synthetase components of the multienzyme complex from sheep liver were selectively dissociated by hydrophobic interaction chromatography on hexyl-agarose and purified to homogeneity. Conservation of activities during the purification required the presence of Triton X-100. The homogeneous enzymes corresponded to a monomer of Mr 129000 and a dimer of Mr 2 X 79000, respectively. Both were strongly adsorbed to the hydrophobic support phenyl-Sepharose, in conditions where the corresponding purified enzymes from yeast and Escherichia coli were not bound. Moreover, like the corresponding enzymes from yeast but unlike those of prokaryotic origin, the purified leucyl-tRNA and lysyl-tRNA synthetases derived from the complex displayed affinity for polyanionic supports. It is shown that proteolytic conversion of lysyl-tRNA synthetase to a fully active dimer of Mr 2 X 64000, leads to loss of both the hydrophobic and the polyanion-binding properties. These results support the view that each subunit of lysyl-tRNA synthetase is composed of a major catalytic domain, similar in size to the subunit of the prokaryotic enzyme, contiguous to a chain extension which carries both cationic charges and hydrophobic residues. The implications of these findings on the structural organization of the complex are discussed in relation to its other known properties.