FUNCTIONAL INTERACTION OF AN ARGININE CONSERVED IN THE 16 AMINO-ACID INSERTION MODULE OF ESCHERICHIA-COLI METHIONYL-TRANSFER-RNA FORMYLTRANSFERASE WITH DETERMINANTS FOR FORMYLATION IN THE INITIATOR TRANSFER-RNA

Formylation of initiator methionyl-tRNA by methionyl-tRNA formyltransf erase (MTF) is important for initiation of protein synthesis in eubact eria. The determinants for formylation are clustered mostly in the acc eptor stem of the initiator tRNA. Previous studies suggested that a 16 amino acid insertion loop, present in all eubacterial MTF's (residues 34-49 in the E. coli enzyme), plays an important role in specific rec ognition of the initiator tRNA. Here, we have analyzed the effect of s ite-specific mutations of amino acids within this region. We show that an invariant arginine at position 42 within the loop plays a very imp ortant role both in the steps of substrate binding and in catalysis. T he kinetic parameters of the R42K and R42L mutant enzymes using accept or stem mutant initiator tRNAs as substrates suggest that arginine 42 makes functional contacts with the determinants at the 3:70 and possib ly also the 2:71 base pairs in the acceptor stem of the initiator tRNA . The kinetic parameters of the G41R/R42L double mutant enzyme are ess entially the same as those of R42L mutant, suggesting that the require ment for arginine at position 42 cannot be fulfilled by an arginine at position 41. Along with other data, this result suggests that the ins ertion loop, which is normally unstructured and flexible, adopts a def ined conformation upon binding to the tRNA.