title: Studies on crenarchaeal tyrosylation accuracy with mutational analyses of tyrosyl-tRNA synthetase and tyrosine tRNA from Aeropyrum pernix.
abstract: Aminoacyl-tRNA synthetases play a key role in the translation of genetic code into correct protein sequences. These enzymes recognize cognate amino acids and tRNAs from noncognate counterparts, and catalyze the formation of aminoacyl-tRNAs. While Although several tyrosyl-tRNA synthetases (TyrRSs) from various species have been structurally and functionally well characterized, the crenarchaeal TyrRS remains poorly understood. In this study, we performed mutational analyses on tyrosine tRNA (tRNA(Tyr)) and TyrRS from the crenarchaeon, Aeropyrum pernix, to investigate the molecular recognition mechanism. Kinetics for tyrosylation using in vitro transcript indicated that the discriminator base A73 and adjacent G72 in the acceptor stem are identity elements of tRNA(Tyr), whereas the C1 base and anticodon had modest roles as identity determinants. Intriguingly, in contrast to the identity element of eukaryotic/euryarchaeal TyrRSs, the first base-pair (C1-G72) of the acceptor stem was not essential in crenarchaeal TyrRS as a pair. Furthermore, A. pernix TyrRS mutants were constructed at positions Tyr39 and Asp172, which could form hydrogen bonds with the 4-hydroxyl group of l-tyrosine. The tyrosylation activities with the mutants resulted that Asp172 mutants completely abolished tyrosylation activity, whereas Tyr39 mutants had no effect on activity. Thus, crenarchaeal TyrRS appears to adopt different molecular recognition mechanism from other TyrRSs.
authors.affiliation: Graduate School of Science and Engineering, Yamagata University, Yamagata, Yamagata 990-8560, Japan.
authors.forename: Jun
authors.initials: J
authors.lastname: Iwaki
authors.forename: Kanako
authors.initials: K
authors.lastname: Endo
authors.forename: Takayuki
authors.initials: T
authors.lastname: Ichikawa
authors.forename: Ryuichiro
authors.initials: R
authors.lastname: Suzuki
authors.forename: Zui
authors.initials: Z
authors.lastname: Fujimoto
authors.forename: Mitsuru
authors.initials: M
authors.lastname: Momma
authors.forename: Atsushi
authors.initials: A
authors.lastname: Kuno
authors.forename: Susumu
authors.initials: S
authors.lastname: Nishimura
authors.forename: Tsunemi
authors.initials: T
authors.lastname: Hasegawa
mesh_terms: D041522:Aeropyrum / Q000201:enzymology* / Q000235:genetics; D019943:Amino Acid Substitution; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D019642:RNA, Archaeal / Q000737:chemistry / Q000235:genetics; D012330:RNA, Double-Stranded / Q000235:genetics; D012365:RNA, Transfer, Tyr / Q000737:chemistry / Q000235:genetics*; D013379:Substrate Specificity; D014158:Transcription, Genetic; D014443:Tyrosine / Q000737:chemistry; D014448:Tyrosine-tRNA Ligase / Q000737:chemistry / Q000235:genetics*
chemical_list: D019843:Archaeal Proteins; D019642:RNA, Archaeal; D012330:RNA, Double-Stranded; D012365:RNA, Transfer, Tyr; D014443:Tyrosine; D014448:Tyrosine-tRNA Ligase