TRYPANOSOMA-BRUCEI U-INSERTION AND U-DELETION ACTIVITIES COPURIFY WITH AN ENZYMATIC EDITING COMPLEX BUT ARE DIFFERENTIALLY OPTIMIZED

RNA editing, the processing that generates functional mRNAs in trypano some mitochondria, involves cycles of protein catalyzed reactions that specifically insert or delete hi residues. We recently reported purif ication from Trypanosoma brucei mitochondria of a complex showing seve n major polypeptides which exhibits the enzymatic activities inferred in editing and that a pool of fractions of the complex catalyzed LB de letion, the minor form of RNA editing in vivo. We now show that U inse rtion activity, the major form of RNA editing in vivo, chromatographic ally co-purifies with both U deletion activity and the protein complex . Furthermore, these editing activities co-sediment at similar to 20 S . U insertion does not require a larger, less characterized complex, a s has been suggested and could have implied that the editing machinery would not function in a processive manner. We also show that U insert ion is optimized at rather different and more exacting reaction condit ions than U deletion. By markedly reducing ATP and carrier RNA and inc reasing UTP and carrier protein relative to standard editing condition s, U insertion activity of the purified fraction is enhanced similar t o 100-fold.