Two acidic residues of Escherichia coli methionyl-tRNA synthetase act as negative discriminants towards the binding of non-cognate tRNA anticodons.
Escherichia coli methionyl-tRNA synthetase recognizes its cognate tRNAs according to the sequence of the CAU anticodon. In order to identify residues of methionyl-tRNA synthetase involved in tRNA anticodon recognition, enzyme variants created by cassette mutagenesis were genetically screened for their acquired ability to charge tRNA(mMet) derivatives with an ochre or an amber anticodon and, consequently, to cause the suppression of a stop codon in an indicator gene. The selected enzymes are called suppressors. Mutations were firstly directed towards the region of the synthetase encompassing residues 451 to 467. Several dozens of suppressor enzymes were compared. Statistical analysis of the mutations suggested that the substitution of an Asp side-chain at position 456 was sufficient to render possible the charging of the ochre or amber suppressor tRNAs. Point mutants at this position were therefore constructed. Their behaviour demonstrated that various tRNA(Met) derivatives having a non-Met anticodon could be aminoacylated in vitro provided only that the side-chain of residue 456 was no longer acidic. In turn, the Asp456 residue is not essential to the CAU anticodon recognition, since its substitution does not impair the aminoacylation of wild-type tRNA(Met). The analysis was enlarged to a second region from residue 437 to residue 454. The mutagenesis highlighted two other positions, one of which, Asn452, appeared involved in wild-type tRNA(Met) binding. The second position, Asp449, plays a role very similar to that of Asp456. It is concluded that both Asp449 and 456 behave as "antideterminants", contributing together to the rejection by the enzyme of tRNAs carrying non-Met anticodons. Finally, it is shown that the activities of some particular methionyl-tRNA synthetase variants, which have been made indifferent to the sequence of the anticodon of a tRNA(Met), are tightly dependent on the presence of the nucleotide determinants specific to the acceptor stem of tRNA(Met).
Schmitt
E
E
Laboratoire de Biochimie, Unité de Recherche Associée no. 240 du Centre National de la Recherche Scientifique Ecole Polytechnique, Palaiseau, France.
Meinnel
T
T
Panvert
M
M
Mechulam
Y
Y
Blanquet
S
S
D000215:Acylation; D000595:Amino Acid Sequence; D000926:Anticodon / Q000378:metabolism*; D001483:Base Sequence; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000201:enzymology*; D008718:Methionine-tRNA Ligase / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012358:RNA, Transfer, Met / Q000378:metabolism*; D015183:Restriction Mapping; D013379:Substrate Specificity
D000926:Anticodon; D004269:DNA, Bacterial; D012358:RNA, Transfer, Met; D008718:Methionine-tRNA Ligase