Nucleotides of tRNA governing the specificity of Escherichia coli methionyl-tRNA fMet formyltransferase

Abstract

In Escherichia coli, the free amino group of the aminoacyl moiety of methionyl-tRNA f Met is specifically modified by a transformylation reaction. To identify the nucleotides governing the recognition of the tRNA substrate by the formylase, initiator tRNA f Met was changed into an elongator tRNA with the help of an in vivo selection method. All the mutations isolated were in the tRNA acceptor arm, at positions 72 and 73. The major role of the acceptor arm was further established by the demonstration of the full formylability of a chimaeric tRNA Met containing the acceptor stem of tRNA f Met and the remaining of the structure of tRNA m Met. In addition, more than 30 variants of the genes encoding tRNA m Met or tRNA f Met have been constructed, the corresponding mutant tRNA products purified and the parameters of the formylation reaction measured. tRNA m Met became formylatable by the only change of the G1 · C72 base-pair into C1-A72. It was possible to render tRNA m Met as good a substrate as tRNA f Met for the formylase by the introduction of a limited number of additional changes in the acceptor stem. In conclusion, A73, G2 · C71, C3 · G70 and G4 · C69 are positive determinants for the specific processing of methionyl-tRNA f Met by the formylase while the occurrence of a G · C or C · G base-pair between positions 1 and 72 acts as a major negative determinant. This pattern appears to account fully for the specificity of the formylase and the lack of formylation of any aminoacylated tRNA, excepting the methionyl-tRNA f Met.