Effects of conserved D/T loop substitutions in the pre-tRNA substrate on tRNase Z catalysis

tRNAs are transcribed as precursors with a 5' end leader and a 3' end trailer. In the course of tRNA maturation, RNase P removes the 5' end leader and tRNase Z can endonucleolytically remove the 3' end trailer. A domain remote from the active site of tRNase Z recognizes and binds substrate, principally through contacts with the elbow (D/T loops) of the tRNA. To evaluate possible contacts, processing kinetics was performed using human nuclear encoded pre-tRNAArg with substitutions in conserved D and T loop nucleotides. Changes in KM observed with some of the substitutions suggest contacts between tRNase Z and substrate tRNA in this region, and changes in tRNA structure provide an additional basis for interpretation of the kinetic effects.