Suppressor mutations in Escherichia coli methionyl-tRNA formyltransferase: role of a 16-amino acid insertion module in initiator tRNA recognition.
The specific formylation of initiator methionyl-tRNA by methionyl-tRNA formyltransferase (MTF; EC 2.1.2.9) is important for the initiation of protein synthesis in eubacteria and in eukaryotic organelles. The determinants for formylation in the tRNA are clustered mostly in the acceptor stem. As part of studies on the molecular mechanism of recognition of the initiator tRNA by MTF, we report here on the isolation and characterization of suppressor mutations in Escherichia coli MTF, which compensate for the formylation defect of a mutant initiator tRNA, lacking a critical determinant in the acceptor stem. We show that the suppressor mutant in MTF has a glycine-41 to arginine change within a 16-amino acid insertion found in MTF from many sources. A mutant with glycine-41 changed to lysine also acts as a suppressor, whereas mutants with changes to aspartic acid, glutamine, and leucine do not. The kinetic parameters of the purified wild-type and mutant Arg-41 and Lys-41 enzymes, determined by using the wild-type and mutant tRNAs as substrates, show that the Arg-41 and Lys-41 mutant enzymes compensate specifically for the strong negative effect of the acceptor stem mutation on formylation. These and other considerations suggest that the 16-amino acid insertion in MTF plays an important role in the specific recognition of the determinants for formylation in the acceptor stem of the initiator tRNA.
Ramesh
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Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.
Gite
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Li
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RajBhandary
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D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D005798:Genes, Bacterial; D019877:Hydroxymethyl and Formyl Transferases / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D012358:RNA, Transfer, Met / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013489:Suppression, Genetic
D012358:RNA, Transfer, Met; D019877:Hydroxymethyl and Formyl Transferases; C021561:methionyl-tRNA formyltransferase