The Interaction Between Leucyl-tRNA Synthetase And TRNA～(Leu)

Leucyl-tRNA synthetase (LeuRS) catalyzes the leucylation of tRNALeu. Tomaintain the fidelity of protein biosynthesis, LeuRS also catalyzes the editingreaction. In the present work, highly conserved T252 in the T-rich region withinCP1 domain of Escherichia coli LeuRS was mutated to G, D, or E. Steady statekinetic of aminoacylation, and combined editing assays indicated that not only thesize of the amino acid but also the absence of hydrogen bonds between T252 andadjacent molecules may affect the editing. It is further confirmed by in vivoexperiments using the temperature-sensitive strain KL231 (?leuS), which revealedthe arrested growth of bacterial cells bearing mutants with highly impaired editingactivity in the presence of leucine analog. We found that the LeuRS-T252E can discriminate two tRNALeu isoacceptors,tRNALeuor tRNALeu. The progress curve of mischarging tRNALeu with isoleucine or 1 2 2methionine by LeuRS-T252E was canonical, however that of mischarging tRNALeu 1showed a burst at the beginning of editing reaction. Accumulation of misaminoacyl-tRNALeu produced by the mutant may be detected in the early stage of the reaction. 1Through mutation of the first base pair in the acceptor stem of the two isoacceptors, - 4 -中国科学院生物化学与细胞生物学研究所博士论文 徐敏刚 摘要it was found that the stabilization of the first base pairing of tRNA determine themisaminoacylation curve. It seems that translocation of mischarged tRNALeu from 1the synthetic active site to the editing active site is slower than that of misaminoacyl-tRNALeu, with the result that misaminoacyl-tRNALeucannot be rapidly deacylated at 2 1the editing active site, leading to its accumulation. Mutation the conserved 76A to Udisables the mutant tRNA from intriguing the editing activity of E. coli LeuRS,however the mutant tRNA still can be aminoacylation efficiently. Meanwhile,LeuRS is the first reported aaRS that can charge a mutant tRNA missing 76A. In a hyperthermophilic bacterium, Aquifex aeolicus, LeuRS consists of twonon-identical polypeptide subunits (α and β), different from the canonical LeuRS,which has a single polypeptide chain. Active site titration assay confirmed that theαβ-LeuRS has only one active site for aminoacylation. The in vitro transcriptionsystem for A. aeolicus tRNALeu was constructed. Aminoacylation and ATP bindingassay confirmed that the β subunit was inactive in catalysis, however it was able tobind tRNALeu, and the competing assay suggested that the β subunit might form twokinds of complexes with tRNA. Aminoacylation of the minihelix mimicking the amino acid acceptor arm oftRNA has been demonstrated in more than ten aminoacyl-tRNA synthetase systems.Although Escherichia coli or Homo sapiens cytoplasmic leucyl-tRNA synthetase(LeuRS) is unable to charge the cognate minihelix or microhelix, we show here thatminihelixLeu is efficiently leucylated by Aquifex aeolicus synthetase, the only knownheterodimeric LeuRS (αβ-LeuRS). Aminoacylation of minihelices is strongly - 5 -中国科学院生物化学与细胞生物学研究所博士论文 徐敏刚 摘要dependent on the presence of the A73 identity nucleotide, and greatly stimulated bydestabilization of the first base pair, as reported for the E. coli isoleucyl-tRNAsynthetase (IleRS) and methionyl-tRNA synthetase (MetRS) systems. In the E. coliLeuRS system, the anticodon of tRNALeu is not important for recognition by thesynthetase. However, the addition of RNA helices mimicking the anticodon domainstimulates minihelixLeu charging by αβ-LeuRS, indicating possible domain-domaincommunication...