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 formyltransfe rase (MTF) is important for the initiation of protein synthesis in eubacter ia. We are studying the molecular mechanisms of recognition of the initiato r tRNA by Escherichia coli MTF. MTF from eubacteria contains an approximate ly 100-amino acid C-terminal extension that is not found in the E. coli gly cinamide ribonucleotide formyltransferase, which, like MTF, use N-10-formyl tetrahydrofolate as a formyl group donor. This C-terminal extension, which forms a distinct structural domain, is attached to the N-terminal domain th rough a Linker region. Here, we describe the effect of (i) substitution mut ations 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 mut ations from the C-terminus on enzyme activity, We show that the positive ch arge on two of the lysine residues in the linker region leading to the C-te rminal domain are important for enzyme activity. Mutation of some of the ba sic amino acids in the C-terminal domain to alanine has mostly small effect s on the kinetic parameters, whereas mutation to glutamic acid has large ef fects. However, the deletion of 18, 20, or 80 amino acids from the C-termin us has very large effects on enzyme activity. Overall, our results support the notion that the basic amino acid residues in the C-terminal domain prov ide a positively charged channel that is used for the nonspecific binding o f tRNA, whereas some of the amino acids in the linker region play an import ant role in activity of MTF.