Critical role of the acceptor stem of tRNAs(Met) in their aminoacylation by Escherichia coli methionyl-tRNA synthetase.
To be aminoacylated by Escherichia coli methionyl-tRNA synthetase, a tRNA requires the presence of the methionine anticodon (CAU sequence). However, the importance in this reaction of the other nucleotides of tRNAs(Met) has still to be described. In this work, through the study of more than 35 variants of tRNAs(Met), it is shown, firstly, that the parameters of the aminoacylation reaction remain independent of the mutations affecting either the sequences or the sizes of the D-loop, D-stem and variable loop. This conclusion is illustrated by the construction and study of a tRNAf(MetCAU) with the D-stem, D-loop and very long variable loop of a class II tRNA. The resulting chimaeric tRNA is methionylated as efficiently as tRNAf(MetCAU) or tRNAm(MetCAU). Secondly, mutations affecting base 73 and base pairs 2.71 and 3.70 in the acceptor stem of tRNAf(MetCAU), as well as bases 32, 33 and 37, adjacent to the anticodon, cause a strong reduction of the rate of the aminoacylation reaction. Thirdly, it is shown that, provided it is given the acceptor stem of tRNAm(MetCAU) or tRNAf(MetCAU), a tRNA having the anticodon loop of tRNA(Met) can be converted into a substrate for methionyl-tRNA synthetase as efficient as tRNAf(MetCAU) or tRNAm(MetCAU). Finally, it is proposed that, beyond the binding of the anticodon loop to the synthetase, the sequence of the acceptor stem may strongly influence the rate of the catalytic step of the aminoacylation reaction by properly orientating the 3'-end of the tRNA towards the catalytic centre.
Meinnel
T
T
Laboratoire de Biochimie, National de la Recherche Scientifique Ecole Polytechnique, Palaiseau, France.
Mechulam
Y
Y
Lazennec
C
C
Blanquet
S
S
Fayat
G
G
D000926:Anticodon; D001483:Base Sequence; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008718:Methionine-tRNA Ligase / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D012358:RNA, Transfer, Met / Q000737:chemistry* / Q000235:genetics
D000926:Anticodon; D012358:RNA, Transfer, Met; D008718:Methionine-tRNA Ligase