The 5' and 3' tRNA processing nucleases from liver mitochondria will be further purified using current methods of enzyme purification. The molecular sizes of the native forms of these two processing nucleases will be measured by sedimentation analysis and moleuclar sieving techniques, and the multiplicity of protein components will be determined by SDS-polyacrylamide gel electrophoresis. Functional studies of the highly purified enzymes will be performed using normal and altered mitochondrial (32P) pre-tRNA transcripts produced in vitro from mtDNA fragments cloned into transcription vectors, or from synthetic templates containing the T7 RNA polymerase promoter. The optimal reaction conditions and spectrum of substrates will be determined using normal mitochondrial tRNA precursor transcripts, and the sequence/structural features of precursor substrates that are critical to recognition and efficient processing will be examined with altered pre-tRNA transcripts produced from chemically synthesized templates. Processed (32P) products will be separated on polyacrylamide/urea gels, excised, and analyzed by conventional fingerprinting using primary and secondary digestions, and end-group analyses. The activity of the 3' processing nuclease will be assayed with (32P) 5'- processed tRNA intermediates. It will be determined whether the mitochondrial 3' processing nuclease is a precise endonuclease, consistent with the activity of the 5' enzyme. The existence of an essential RNA component associated with the 5' and 3' processing nucleases will be tested by sensitivity of these enzymes to micrococcal nuclease digestion. The RNA component(s) will be isolated and sequenced by current RNA sequencing proteocols to determine the probable site of origin (mitochondrial or nuclear) of the RNA(s). In addition, the cytosolic (extra-mitochondrial) 5' processing nuclease will be further purified for direct comparison with the mitochondrial counterpart.
