MATRICES OF PAIRED SUBSTITUTIONS SHOW THE EFFECTS OF TRANSFER-RNA D TLOOP SEQUENCE ON DROSOPHILA RNASE-P AND 3'-TRNASE PROCESSING/

Drosophila RNase P and 3'-tRNase endonucleolytically process the 5' an d 3' ends of tRNA precursors, We examined the processing kinetics of n ormal substrates and the inhibitory effect of the tRNA product on both processing reactions. The product is not a good RNase P inhibitor, wi th a K-I, approximately 7 times greater than the substrate K-M Of simi lar to 200 nM and is a better inhibitor of 3'-tRNase, with a K-I, appr oximately two times the K-M, of similar to 80 nM. We generated matrice s of substitutions at positions G(18)/U-55 and G(19)/C-56 (two contigu ous universally conserved D/T loop base pairs) in Drosophila tRNA prec ursors, More than half the variants display a significant reduction in their ability to be processed by RNase P and 3'-tRNase, Minimal subst rates with deleted D and anticodon stems could be processed by RNase P and 3'-tRNase much like full-length substrates, indicating that D/T l oop contacts and D arm/enzyme contacts are not required by either enzy me. Selected tRNAs that were poor substrates for one or both enzymes w ere further analyzed using Michaelis-Menten kinetics and by structure probing, Processing reductions arise principally due to an increase in K-M, with relatively little change in V-max,,, consistent with the re mote location of the sequence and structure changes from the processin g site for both enzymes. Local changes in variant tRNA susceptibility to RNase T1 and RNase A did not coincide with processing disabilities.