A conserved threonine within Escherichia coli leucyl-tRNA synthetase prevents hydrolytic editing of leucyl-tRNALeu.
Aminoacyl-tRNA synthetases ensure the fidelity of protein synthesis by accurately selecting and activating cognate amino acids for aminoacylation of the correct tRNA. Some tRNA synthetases have evolved an editing active site that is separate from the amino acid activation site providing two steps or "sieves" for amino acid selection. These two sieves rely on different strategies for amino acid recognition to significantly enhance the accuracy of aminoacylation. We have performed alanine scanning mutagenesis in a conserved threonine-rich region of the Escherichia coli leucyl-tRNA synthetase's CP1 domain that is hypothesized to contain a putative editing active site. Characterization of purified mutant proteins led to the identification of a single conserved threonine that prevents the cognate leucine amino acid from being hydrolyzed after aminoacylation of the tRNA. Mutation of this threonine to an alanine eliminates discrimination of the cognate amino acid in the editing active site. This provides a molecular example of an amino acid discrimination mechanism in the tRNA synthetase's editing active site.
Mursinna
R S
RS
Department of Biology and Biochemistry, University of Houston, Texas 77204-5513, USA.
Lincecum
T L
TL
Martinis
S A
SA
D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D017124:Conserved Sequence; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006868:Hydrolysis; D007935:Leucine-tRNA Ligase / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary / Q000235:genetics; D017393:RNA Editing / Q000235:genetics; D012356:RNA, Transfer, Leu / Q000235:genetics / Q000378:metabolism*; D013912:Threonine / Q000235:genetics / Q000378:metabolism*; D046249:Transfer RNA Aminoacylation / Q000235:genetics
D012356:RNA, Transfer, Leu; D013912:Threonine; D007935:Leucine-tRNA Ligase; D000409:Alanine