Induced fit of a peptide loop of methionyl-tRNA formyltransferase triggered by the initiator tRNA substrate

A 16-aa insertion loop present in eubacterial methionyl-tRNA formyltransfer ases (MTF) is critical for specific recognition of the initiator tRNA in Es cherichia coli, We have studied the interactions between this region of the E, coli enzyme and initiator methionyl-tRNA (Met-tRNA) by using two comple mentary protection experiments: protection of MTF against proteolytic cleav age by tRNA and protection of tRNA against nucleolytic cleavage by MTF, The insertion loop in MTF is uniquely sensitive to cleavage by trypsin. We sho w that the substrate initiator Met-tRNA protects MTF against trypsin cleava ge, whereas a formylation-defective mutant initiator Met-tRNA, which binds to MTF with approximately the same affinity, does not. Also, mutants of MTF within the insertion loop (which are defective in formylation) are not pro tected by the initiator Met-tRNA, Thus, a functional enzyme-substrate compl ex is necessary for protection of MTF against trypsin cleavage. Along with other data, these results strongly suggest that a segment of the insertion loop, which is exposed and unstructured in MTF, undergoes an induced fit in the functional MTF-Met-tRNA complex but not in the nonfunctional one. Foot printing experiments show that MTF specifically protects the acceptor stem and the 3'-end region of the initiator Met-tRNA against cleavage by double and single strand-specific nucleases, This protection also depends on forma tion of a functional MTF Met-tRNA complex. Thus, the insertion loop interac ts mostly with the acceptor stem of the initiator Met-tRNA, which contains the critical determinants for formylation.