Effect of changes in the flexible arm on tRNase Z processing kinetics

Abstract

tRNAs are transcribed as precursors and processed in a series of reactions culminating in aminoacylation and translation. Central to tRNA maturation, the 3′ end trailer can be endonucleolytically removed by tRNase Z. A flexible arm (FA) extruded from the body of tRNase Z consists of a structured ααββ hand that binds the elbow of pre-tRNA. Deleting the FA hand causes an almost 100-fold increase in Km with little change in kcat, establishing its contribution to substrate recognition/binding. Remarkably, a 40-residue Ala scan through the FA hand reveals a conserved leucine at the ascending stalk/hand boundary that causes practically the same increase in Km as the hand deletion, thus nearly eliminating its ability to bind substrate. Km also increases with substitutions in the GP(α4-α5) loop and at other conserved residues in the FA hand predicted to contact substrate based on the co-crystal structure. Substitutions that reduce kcat are clustered in the β10-β11 loop. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.