Threonyl-tRNA synthetase (ThrRS), which catalyzes the threonylation reaction of attaching Thr to tRNA^Thr, is essential for protein synthesis in all cells. The research group led by Prof. WANG Enduo from the Institute of Biochemistry and Cell Biology (SIBCB), Shanghai Institutes for Biological Sciences of Chinese Academy of Sciences, performed a genetic identification on the functional features of yeast cytoplasmic ThrRS (ScThrRS). Their research aimed to capture important residues of eukaryotic ThrRSs in the dynamic process of threonylation and editing.

ThrRS belongs to class II aminoacyl-tRNA synthetase and contains an antiparallel β-fold catalytic center with three motifs: motif 1, 2 and 3. At present, the active site of bacterial ThrRS has been studied extensively, but little research has been done on eukaryotic ThrRS. For instance, eukaryotic ThrRS has evolved a unique eukaryotic-specific N-terminal extension whose relevance in aminoacylation and editing is not completely understood.

Increasing evidence suggests that these essential enzymes are often associated with human disorders. Therefore, complete determination of the functions of eukaryotic ThrRS not only uncovers the catalytic mechanism of the enzyme, but also is essential for gaining insights into the pathological mechanisms induced by human ThrRS mutations.

Under the guidance of Prof. WANG Enduo and Dr. ZHOU Xiaolong, graduate students RUAN Zhirong et al identified 12 loss-of-function mutations of ScThrRS by using a genetic selection in the ScThrRS gene knockout strain. The mutations are spread over the four main functional domains of ScThrRS, indicating that the selection procedure could map the critical residues and enzyme functions efficiently. They showed that the selected residues affected activities of ScThrRS in various manners and the N-terminal extension was important in maintaining protein structure and function. Introducing the same mutations into human cytoplasmic ThrRS (hcThrRS), they found that the complement phenotypes and protein amounts of hcThrRS mutants was similar to analogous ScThrRS mutants.

These results highlight the synthetic and editing functions of yeast and human ThrRS, and open perspectives on studies on the pathology-associated hcThrRS mutants in the future.

This work entitled "Identification of lethal mutations in yeast threonyl-tRNA synthetase which reveals critical residues in its human homolog" was published on Journal of Biological Chemistry on November 26, 2014.

The study was supported by grants from the National Natural Science Foundation of China, Ministry of Science and Technology of China, Chinese Academy of Sciences and Science and Technology Commission of Shanghai Municipality. 

Figure: Identification of lethal mutations in yeast threonyl-tRNA synthetase. (A) Schematic representation of the genetic screening procedure used to select mutated yeast ThrRS gene library. (B) Selected mutated residues are shown on the crystallographic structure of E. coli ThrRS-tRNA^Thr complex. (Image by Prof. WANG Enduo's group.)