Functional analyses for tRNase Z variants: An aspartate and a histidine in the active site are essential for the catalytic activity

Abstract

We performed functional analyses for various single amino-acid substitution variants of Escherichia coli, Bacillus subtilis, and human tRNase Zs. The well-conserved six histidine, His(I)–His(VI), and two aspartate, Asp(I) and Asp(II), residues together with metal ions are thought to form the active site of tRNase Z. The Mn 2+-rescue analysis for Thermotoga maritima tRNase Z S has suggested that Asp(I) and His(V) directly contribute the proton transfer for the catalysis, and a catalytic mechanism has been proposed. However, experimental evidence supporting the proposed mechanism was limited. Here we intensively examined E. coli and B. subtilis tRNase Z S variants and human tRNase Z L variants for cleavage activities on pre-tRNAs in the presence of Mg 2+ or Mn 2+ ions. We observed that the Mn 2+ ions cannot rescue the activities of Asp(I)Ala and His(V)Ala variants from each species, which are lost in the presence of Mg 2+. This observation may support the proposed catalytic mechanism.