Matrices of paired substitutions show the effects of tRNA D/T loop sequence on Drosophila RNase P and 3′-tRNase processing

Drosophila RNase P and 3'-tRNase endonucleolytically process the 5' and 3' ends of tRNA precursors, We examined the processing kinetics of normal substrates and the inhibitory effect of the tRNA product on both processing reactions. The product is not a good RNase P inhibitor, with a K-I, approximately 7 times greater than the substrate K-M Of similar to 200 nM and is a better inhibitor of 3'-tRNase, with a K-I, approximately two times the K-M, of similar to 80 nM. We generated matrices of substitutions at positions G(18)/U-55 and G(19)/C-56 (two contiguous universally conserved D/T loop base pairs) in Drosophila tRNA precursors, More than half the variants display a significant reduction in their ability to be processed by RNase P and 3'-tRNase, Minimal substrates with deleted D and anticodon stems could be processed by RNase P and 3'-tRNase much like full-length substrates, indicating that D/T loop contacts and D arm/enzyme contacts are not required by either enzyme. Selected tRNAs that were poor substrates for one or both enzymes were 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 remote location of the sequence and structure changes from the processing site for both enzymes. Local changes in variant tRNA susceptibility to RNase T1 and RNase A did not coincide with processing disabilities.