L. Levinger et al., MATRICES OF PAIRED SUBSTITUTIONS SHOW THE EFFECTS OF TRANSFER-RNA D TLOOP SEQUENCE ON DROSOPHILA RNASE-P AND 3'-TRNASE PROCESSING/, The Journal of biological chemistry, 273(2), 1998, pp. 1015-1025
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.