A site in the dinucleotide-fold domain contributes to the accuracy of tRNA selection by Escherichia coli methionyl-tRNA synthetase.
Interactions of specific amino acid residues of the carboxyl-terminal domain of MetRS with the CAU anticodon of tRNAMet assure accurate and efficient aminoacylation. The substitution of one such residue, Trp461 by Phe, impairs the binding of cognate tRNA, but enhances the binding of noncognate tRNAs, particularly those containing G at the wobble position. However, the enhanced binding of noncognate tRNAs is not accompanied by the increased aminoacylation of these tRNAs. A genetic screening procedure was designed to isolate methionyl-tRNA synthetase mutants which were able to aminoacylate a GGU (threonine) anticodon derivative of tRNAfMet. One such mutant, obtained from W461F MetRS, had an Ile29 to Thr substitution in helix A located in the amino-terminal dinucleotide-fold domain that forms the site for amino acid activation. Analysis of the catalytic properties of the I29T/W461F enzyme indicates that the mutation in helix A of the dinucleotide-fold domain affects kcat for aminoacylation of tRNAs having a GGU threonine anticodon. Interactions with cognate tRNAfMet (CAU), as well as with methionine and ATP were not affected by the Ile29 to Thr substitution. We conclude that the I29T substitution leads to a slight adjustment of the alignment of the CCA stem of noncognate tRNAs (GGU) in the catalytic domain of the enzyme, reflected in the increase in kcat, which also allows mischarging in vivo. A function of Ile29 is therefore to minimize the mischarging of tRNAThr (GGU) by methionyl-tRNA synthetase. The methods described here provide useful tools for examining the mechanisms of tRNA selection by aminoacyl-tRNA synthetases.
Kim
H Y
HY
Department of Food Science and Institute of Genetic Engineering, Kyung-Hee University, Suwon, Korea. hykim@nms.kyunghee.ac.kr
Pak
M
M
Jakubowski
H
H
D000926:Anticodon / Q000235:genetics; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D014644:Genetic Variation; D008718:Methionine-tRNA Ligase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry / Q000235:genetics*; D012346:RNA, Transfer, Amino Acyl / Q000235:genetics / Q000378:metabolism*; D012358:RNA, Transfer, Met / Q000235:genetics / Q000378:metabolism
D000926:Anticodon; D009841:Oligonucleotides; D012346:RNA, Transfer, Amino Acyl; D012358:RNA, Transfer, Met; D008718:Methionine-tRNA Ligase