Residues in two homology blocks on the amino side of the tRNase Z His domain contribute unexpectedly to pre-tRNA 3′ end processing

Abstract

tRNase Z, which can endonucleolytically remove pre-tRNA 3′-end trailers, possesses the signature His domain (HxHxDH; Motif II) of the β-lactamase family of metal-dependent hydrolases. Motif II combines with Motifs III–V on its carboxy side to coordinate two divalent metal ions, constituting the catalytic core. The PxKxRN loop and Motif I on the amino side of Motif II have been suggested to modulate tRNase Z activity, including the anti-determinant effect of CCA in mature tRNA. Ala walks through these two homology blocks reveal residues in which the substitutions unexpectedly reduce catalytic efficiency. While substitutions in Motif II can drastically affect kcat without affecting kM, five- to 15-fold increases in kM are observed with substitutions in several conserved residues in the PxKxRN loop and Motif I. These increases in kM suggest a model for substrate binding. Expressed tRNase Z processes mature tRNA with CCA at the 3′ end ∼80 times less efficiently than a pre-tRNA possessing natural sequence of the 3′-end trailer, due to reduced kcat with no effect on kM, showing the CCA anti-determinant to be a characteristic of this enzyme.