title: The determination of tRNALeu recognition nucleotides for Escherichia coli L/F transferase.
abstract: Escherichia coli leucyl/phenylalanyl-tRNA protein transferase catalyzes the tRNA-dependent post-translational addition of amino acids onto the N-terminus of a protein polypeptide substrate. Based on biochemical and structural studies, the current tRNA recognition model by L/F transferase involves the identity of the 3' aminoacyl adenosine and the sequence-independent docking of the D-stem of an aminoacyl-tRNA to the positively charged cluster on L/F transferase. However, this model does not explain the isoacceptor preference observed 40 yr ago. Using in vitro-transcribed tRNA and quantitative MALDI-ToF MS enzyme activity assays, we have confirmed that, indeed, there is a strong preference for the most abundant leucyl-tRNA, tRNA(Leu) (anticodon 5'-CAG-3') isoacceptor for L/F transferase activity. We further investigate the molecular mechanism for this preference using hybrid tRNA constructs. We identified two independent sequence elements in the acceptor stem of tRNA(Leu) (CAG)-a G₃:C₇₀ base pair and a set of 4 nt (C₇₂, A₄:U₆₉, C₆₈)-that are important for the optimal binding and catalysis by L/F transferase. This maps a more specific, sequence-dependent tRNA recognition model of L/F transferase than previously proposed.
authors.affiliation: Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.
authors.forename: Angela Wai Shan
authors.initials: AW
authors.lastname: Fung
authors.affiliation: Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.
authors.forename: Charles Chung Yun
authors.initials: CC
authors.lastname: Leung
authors.affiliation: Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7 Department of Oncology, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.
authors.forename: Richard Peter
authors.initials: RP
authors.lastname: Fahlman
mesh_terms: D019881:Aminoacyltransferases / Q000378:metabolism*; D000926:Anticodon; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D009690:Nucleic Acid Conformation; D009711:Nucleotides; D012346:RNA, Transfer, Amino Acyl; D012356:RNA, Transfer, Leu / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D013379:Substrate Specificity; D046249:Transfer RNA Aminoacylation
chemical_list: D000926:Anticodon; D009711:Nucleotides; D012346:RNA, Transfer, Amino Acyl; D012356:RNA, Transfer, Leu; D019881:Aminoacyltransferases; C021511:leucyltransferase
keywords: L/F transferase; N-end rule; aminoacyl-tRNA protein transferase; isoacceptors; quantitative mass spectrometry; tRNA recognition