Activation of tRNA Maturation by Downstream Uracil Residues in B. subtilis

Ribonuclease (RNase) Z is involved in the maturation of the 3′ ends of transfer RNAs (tRNAs) in all three kingdoms of life. To prevent futile cycles of CCA addition and removal, eukaryotic RNase Z discriminates against mature tRNAs bearing a CCA motif, with the first cytosine residue (C74) being the key antideterminant. Here, we show that, remarkably, the B. subtilis enzyme does not discriminate against cytosine in position 74, but rather is highly stimulated by uracil in this location. Consistent with this observation, the vast majority of B. subtilis tRNA precursor substrates of RNase Z naturally contain U74. Those tRNA precursors with a uracil further downstream are also substrates for RNase Z, but are matured in a two-step endo/exonuclease reaction. We solved the first crystal structure of B. subtilis RNase Z bound to a tRNAThr precursor with U74 and show that the enzyme has a specific binding pocket for this nucleotide. ► B. subtilis RNase Z endonuclease activity is stimulated by downstream uracil ► Most B. subtilis tRNAs naturally have uracil in position 74 ► tRNAs with U75 or U76 are matured in two-step endo/exonucleolytic mechanism ► Structure of RNase Z bound to substrate identifies specific uracil binding pocket To prevent futile cycles of CCA addition and removal at the 3′ ends of tRNAs, eukaryotic RNase Z discriminates against mature tRNAs, with the first C of the CCA motif being critical. Pellegrini et al. show that, instead of discriminating against the C, the B. subtilis enzyme is highly stimulated by U at this location.