Accurate tRNA 3¢ end maturation is essential for aminoacylation and thus for protein synthesis in all organisms. Here we report the ®rst identi®cation of protein and DNA sequences for tRNA 3¢-processing endonucleases (RNase Z). Puri®cation of RNase Z from wheat identi®ed a 43 kDa protein correlated with the activity. Peptide sequences obtained from the puri®ed protein were used to identify the corresponding gene. In vitro expression of the homologous proteins from Arabidopsis thaliana and Methanococcus janaschii con®rmed their tRNA 3¢-processing activities. These RNase Z proteins belong to the ELAC1/2 family of proteins and to the cluster of orthologous proteins COG 1234. The RNase Z enzymes from A.thaliana and M.janaschii are the ®rst members of these families to which a function can now be assigned. Proteins with high sequence similarity to the RNase Z enzymes from A.thaliana and M.janaschii are present in all three kingdoms.
One of the essential maturation steps to yield functional tRNA molecules is the removal of 3'-trailer sequences by RNase Z. After RNase Z cleavage the tRNA nucleotidyl transferase adds the CCA sequence to the tRNA 3'-terminus, thereby generating the mature tRNA. Here we investigated whether a terminal CCA triplet as 3'-trailer or embedded in a longer 3'-trailer influences cleavage site selection by RNase Z using three activities: a recombinant plant RNase Z, a recombinant archaeal RNase Z and an RNase Z active wheat extract. A trailer of only the CCA trinucleotide is left intact by the wheat extract RNase Z but is removed by the recombinant plant and archaeal enzymes. Thus the CCA triplet is not recognized by the RNase Z enzyme itself, but rather requires cofactors still present in the extract. In addition, we investigated the influence of acceptor stem length on cleavage by RNase Z using variants of wild-type tRNATyr. While the wild type and the variant with 8 base pairs in the acceptor stem were processed efficiently by all three activities, variants with shorter and longer acceptor stems were poor substrates or were not cleaved at all.
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