Importance of 3-CCA End of tRNA^Leu to Quality Control of Protein Biosynthesis

Protein synthesis is a central process in organisms from all three domains of life, providing the link between the genetic information encoded in DNA and functional proteins. This process critically relies on the correct formation of aminoacyl-tRNA (aa-tRNA) by aminoacyl-tRNA synthetase (aaRS) to produce correct materials of protein synthesis. Accurate recognition of aaRS to its cognate amino acid and tRNA to form correct aa-tRNA ensures the fidelity of protein biosynthesis. Mis-translation arising from disruption in the fidelity of these interactions has severe consequences to cells. However, the presence of various types of amino acids and their analogues, and the fact that amino acids only differ in the side chain, greatly challenge the specificity of aaRS. About half of the aaRSs misactivate non-cognate amino acids and mischarge their cognate tRNA with the non-cognate amino acids. To solve this problem, the proofreading (editing) mechanism of aaRS has evolved to hydrolyze the mis-intermediate and mis-products. Editing can occur at the aa-AMP level (pre-transfer editing) and/or misaminoacyl-tRNA level (post-transfer editing), depending on the various aaRSs. The CP1 domain of leucyl-tRNA synthetase (LeuRS) is located 35 Å away from its synthetic domain, Rossmann fold domain, and is responsible for post-transfer editing. Recent studies in WANG Enduo’s lab have revealed that LeuRSs from Escherichia coli, Aquifex aeolicus and human cytoplasm (EcLeuRS, AaLeuRS and hcLeuRS) all have three different editing pathways (tRNA-independent, tRNA-dependent pre-transfer editing, and post-transfer editing), however they prefer various editing pathway to remove non-cognate amino acids. The role of tRNA^Leu and its interacting sites in the CP1 domain of LeuRS on the fidelity of protein synthesis is undefined.

 

Recently, ZHOU Xiaolong and his colleagues led by WANG Enduo at the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, CAS studied the role of tRNA^Leu of Escherichia coli on the accurate recognition by LeuRS. Using in vivo and in vitro methods, they investigated the effect of nucleotide mutagenesis in the CCA₇₆ accepting end of tRNA^Leu on the aminoacylation and editing reactions. The results showed that the terminal A₇₆ is essential for both reactions, suggesting that the critical interactions between tRNA and LeuRS occur in the two catalytic active sites. Substitutions of C₇₄ and C₇₅ selectively decreased aminoacylation without any effect to editing. The tRNA^Leu A₇₆-deficient mutant was a substrate of EcLeuRS in aminoacylation but failed to stimulate editing. Mutating one or more residues along the conserved CCA₇₆ entrance pathway significantly impacted aminoacylation fidelity, leading to cytotoxicity with non-cognate amino acids. Furthermore, different tRNA^Leu iso-acceptors had identical editing-stimulating capacity, which was partly regulated by the 1st base-pair pattern. The results illustrates how protein synthesis quality is controlled and reinforces the significance of CCA₇₆ homogeneity of tRNAs.

 

This work entitled “Role of tRNA amino acid-accepting end in aminoacylation and its quality control” was published in Nucleic Acids Research on July 20, 2011.

 

This work was supported by the Natural Science Foundation of China, Committee of Science and Technology in Shanghai, a Visiting Professorship for Senior International Scientists from the Chinese Academy of Sciences and Programme International de Coopération Scientifique from CNRS.

 

AUTHOR CONTACT:

WANG Enduo

Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China

Phone:86-21-54921241; E-mail: edwang@sibs.ac.cn