Molecular mechanism of tRNA binding by theEscherichia coliN7 guanosine methyltransferase TrmB

Among the large and diverse collection of tRNA modifications, 7-methylguanosine is frequently found in the tRNA variable loop at position 46. This modification is introduced by the TrmB enzyme, which is conserved in bacteria and eukaryotes. Complementing the report of various phenotypes for different organisms lacking TrmB homologs, we report here hydrogen peroxide sensitivity for the Escherichia coli ΔtrmB knockout strain. To gain insight into the molecular mechanism of tRNA binding by E. coli TrmB in real-time, we developed a new assay based on introducing a 4-thiouridine modification at position 8 of in vitro transcribed tRNA Phe enabling us to fluorescently labelled this unmodified tRNA. Using rapid kinetic stopped-flow measurements with this fluorescent tRNA, we examined the interaction of wild-type and single substitution variants of TrmB with tRNA. Our results reveal the role of SAM for rapid and stable tRNA binding, the rate-limiting nature of m 7 G46 catalysis for tRNA release, and the importance of residues R26, T127 and R155 across the entire surface of TrmB for tRNA binding.