Escherichia coli methionyl-tRNA formyltransferase: role of amino acids conserved in the linker region and in the C-terminal domain on the specific recognition of the initiator tRNA.
The formylation of initiator methionyl-tRNA by methionyl-tRNA formyltransferase (MTF) is important for the initiation of protein synthesis in eubacteria. We are studying the molecular mechanisms of recognition of the initiator tRNA by Escherichia coli MTF. MTF from eubacteria contains an approximately 100-amino acid C-terminal extension that is not found in the E. coli glycinamide ribonucleotide formyltransferase, which, like MTF, use N(10)-formyltetrahydrofolate as a formyl group donor. This C-terminal extension, which forms a distinct structural domain, is attached to the N-terminal domain through a linker region. Here, we describe the effect of (i) substitution mutations on some nineteen basic, aromatic and other conserved amino acids in the linker region and in the C-terminal domain of MTF and (ii) deletion mutations from the C-terminus on enzyme activity. We show that the positive charge on two of the lysine residues in the linker region leading to the C-terminal domain are important for enzyme activity. Mutation of some of the basic amino acids in the C-terminal domain to alanine has mostly small effects on the kinetic parameters, whereas mutation to glutamic acid has large effects. However, the deletion of 18, 20, or 80 amino acids from the C-terminus has very large effects on enzyme activity. Overall, our results support the notion that the basic amino acid residues in the C-terminal domain provide a positively charged channel that is used for the nonspecific binding of tRNA, whereas some of the amino acids in the linker region play an important role in activity of MTF.
Gite
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Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
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Ramesh
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RajBhandary
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D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D017124:Conserved Sequence; D004789:Enzyme Activation / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D019877:Hydroxymethyl and Formyl Transferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010442:Peptide Chain Initiation, Translational / Q000235:genetics; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D012358:RNA, Transfer, Met / Q000378:metabolism*; D017384:Sequence Deletion
D010446:Peptide Fragments; D012358:RNA, Transfer, Met; C024988:tRNA, formylmethionine-; D019877:Hydroxymethyl and Formyl Transferases; C021561:methionyl-tRNA formyltransferase