Cleavage Specificity of Chloroplast and Nuclear tRNA 3′-Processing Nucleases

Abstract

tRNAs in eukaryotic nuclei and organelles are synthesized as precursors lacking the 3′-terminal CCA sequence and possessing 5′ (leader) and 3′ (trailer) extensions. Nucleolytic cleavage of the 3′ trailer and addition of CCA are therefore required for formation of functional tRNA 3′ termini. Many chloroplast tRNA genes encode a C at position 74 which is not removed during processing but which can be incorporated as the first base of the CCA_OH terminus. Sequences downstream of nucleotide 74, however, are always removed. Synthetic yeast pre-tRNA^Phe substrates containing the complete CCA₇₄_₇₆ sequence were processed with crude or partially purified chloroplast enzyme fractions. The 3′-extended substrates (tRNA-CCA-trailer) were cleaved exclusively between nucleotides 74 and 75 to give tRNA-C_OHI, whereas a 3′-mature transcript (tRNA-CCA_OH) was not cleaved at all. A 5′-, 3′-extended chloroplast tRNA-CAG-trailer was also processed entirely to tRNA-C_OH. Furthermore, a 5′-mature, 3′-extended yeast pre-tRNA^Phe derivative, tRNA-ACA- trailer, in which C₇₄ was replaced by A, was cleaved precisely after A₇₄. In contrast, we found that a partially purified enzyme fraction (a nuclear/cytoplasmic activity) from wheat embryo cleaved the 3′-extended yeast tRNA^Phe precursors between nucleotides 73 and 74 to give tRNA_OH. This specificity is consistent with that of all previously characterized nuclear enzyme preparations. We conclude that (i) chloroplast tRNA 3′-processing endonuclease cleaves after nucleotide 74 regardless of the nature of the surrounding sequences; (ii) this specificity differs from that of the plant nuclear/cytoplasmic processing nuclease, which cleaves after base 73; and (iii) since 3′-mature tRNA is not a substrate for either activity, these 3′ nucleases must require substrates possessing a 3′-terminal extension that extends past nucleotide 76. This substrate specificity may prevent mature tRNA from counterproductive cleavage by the 3′ processing system.