Substitutions in conserved regions preceding and within the linker affect activity and flexibility of tRNase Z(L), the long form of tRNase Z

The enzyme tRNase Z, a member of the metallo-β-lactamase family, endonucleolytically removes 3’ trailers from precursor tRNAs, preparing them for CCA addition and aminoacylation. The short form of tRNase Z, tRNase Z(S), functions as a homodimer and is found in all prokaryotes and some eukaryotes. The long form, tRNase Z(L), related to tRNase Z(S) through tandem duplication and found only in eukaryotes, possesses ~2,000-fold greater catalytic efficiency than tRNase Z(S). tRNase Z(L) consists of related but diverged amino and carboxy domains connected by a flexible linker (also referred to as a flexible tether) and functions as a monomer. The amino domain retains the flexible arm responsible for substrate recognition and binding while the carboxy domain retains the active site. The linker region was explored by Ala-scanning through two conserved regions of D. melanogaster tRNase Z: N(dom)T(prox), located at the carboxy end of the amino domain proximal to the linker, and T(flex), a flexible site in the linker. Periodic substitutions in a hydrophobic patch (F(329) and L(332)) at the carboxy end of N(dom)T(prox) show 2,700 and 670-fold impairment relative to wild type, respectively, accompanied by reduced linker flexibility at N-T inside the N(dom)- linker boundary. The Ala substitution for N(378) in the T(flex) region has 10-fold higher catalytic efficiency than wild type and locally decreased flexibility, while the Ala substitution at R(382) reduces catalytic efficiency ~50-fold. These changes in pre-tRNA processing kinetics and protein flexibility are interpreted in light of a recent crystal structure for S. cerevisiae tRNase Z, suggesting transmission of local changes in hydrophobicity into the skeleton of the amino domain.