Separation and characterization of 5'- and 3'-tRNA processing nucleases from rat liver mitochondria.

The 5'- and 3'-tRNA processing nucleases have been isolated from rat liver mitochondria. The two activities co-purified through heparin-agarose and phenyl-Sepharose columns and then efficiently separated on a DEAE-cellulose column. The 5' processing nuclease was found in the flow-through fraction, and the 3' processing activity eluted with 0.5 M KCl. Both enzymes were greater than 500-fold purified over the high speed supernatant of a mitoplast extract. The 159-base pre-tRNATyr used as a substrate in this study was synthesized in vitro and contained the Escherichia coli suppressor III tRNATyr plus a 49-base leader sequence and a 25-base trailing sequence. The 5' processing nuclease converted the pre-tRNATyr into two discrete RNA species, identified as the 5'-processed intermediate and the 5' flanking fragment, by endonucleolytic cleavage at the 5' end of the mature tRNATyr sequence. The 3' processing nuclease was inactive with the intact pre-tRNATyr as substrate but efficiently converted the 5'-processed intermediate to the mature tRNATyr, indicating an obligatory order of processing in which 5' maturation was necessary before cleavage by the 3' processing nuclease could occur. The mitochondrial enzymes exhibited optimal activity in the presence of about 2 mM Mg2+, but both enzymes were nearly fully active without addition of exogenous Mg2+ to the reaction mixtures. In contrast, a partially purified 5' processing endonuclease present in the postmitochondrial cytosolic fraction required higher [Mg2+] for activity, thus providing a means for differentiating between these similar enzyme activities obtained from the cytosolic and mitochondrial fractions.