title: The Yin and Yang of tRNA: proper binding of acceptor end determines the catalytic balance of editing and aminoacylation.
abstract: Faithful translation of the genetic code depends on accurate coupling of amino acids with cognate transfer RNAs (tRNAs) catalyzed by aminoacyl-tRNA synthetases. The fidelity of leucyl-tRNA synthetase (LeuRS) depends mainly on proofreading at the pre- and post-transfer levels. During the catalytic cycle, the tRNA CCA-tail shuttles between the synthetic and editing domains to accomplish the aminoacylation and editing reactions. Previously, we showed that the Y330D mutation of Escherichia coli LeuRS, which blocks the entry of the tRNA CCA-tail into the connective polypeptide 1 domain, abolishes both tRNA-dependent pre- and post-transfer editing. In this study, we identified the counterpart substitutions, which constrain the tRNA acceptor stem binding within the synthetic active site. These mutations negatively impact the tRNA charging activity while retaining the capacity to activate the amino acid. Interestingly, the mutated LeuRSs exhibit increased global editing activity in the presence of a non-cognate amino acid. We used a reaction mimicking post-transfer editing to show that these mutations decrease post-transfer editing owing to reduced tRNA aminoacylation activity. This implied that the increased editing activity originates from tRNA-dependent pre-transfer editing. These results, together with our previous work, provide a comprehensive assessment of how intra-molecular translocation of the tRNA CCA-tail balances the aminoacylation and editing activities of LeuRS.
authors.affiliation: Center for RNA research, State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, The Chinese Academy of Sciences, Shanghai 200031, PR China.
authors.forename: Min
authors.initials: M
authors.lastname: Tan
authors.forename: Meng
authors.initials: M
authors.lastname: Wang
authors.forename: Xiao-Long
authors.initials: XL
authors.lastname: Zhou
authors.forename: Wei
authors.initials: W
authors.lastname: Yan
authors.forename: Gilbert
authors.initials: G
authors.lastname: Eriani
authors.forename: En-Duo
authors.initials: ED
authors.lastname: Wang
mesh_terms: D019943:Amino Acid Substitution; D055162:Biocatalysis; D020134:Catalytic Domain; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007935:Leucine-tRNA Ligase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D017434:Protein Structure, Tertiary; D017393:RNA Editing; D012343:RNA, Transfer / Q000737:chemistry / Q000378:metabolism; D046249:Transfer RNA Aminoacylation
chemical_list: D029968:Escherichia coli Proteins; D012343:RNA, Transfer; D007935:Leucine-tRNA Ligase