Copyright © 1992, American Society for Microbiology Cleavage Specificity of Chloroplast and Nuclear tRNA

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 CCAOH terminus. Sequences downstream of nucleotide 74, however, are always removed. Synthetic yeast pre-tRNA"te substrates containing the complete CCA7476 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-COH, whereas a 3'-mature transcript (tRNA-CCAoH) was not cleaved at all. A 5'-, 3'-extended chloroplast tRNA-CAG-trailer was also processed entirely to tRNA-COH. Furthermore, a 5'-mature, 3'-extended yeast pre-tRNAPhe 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 tRNAPhe precursors between nucleotides 73 and 74 to give tRNAOH. 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) thi