INTERACTION BETWEEN ESCHERICHIA-COLI RNASE-P RNA AND THE DISCRIMINATOR BASE RESULTS IN SLOW PRODUCT RELEASE

We suggested previously that a purine at the discriminator base positi on ina tRNA precursor interacts with the well-conserved U294 in M1 RNA , the catalytic subunit of Escherichia coli RNase P. Here we investiga ted this interaction and its influence on the kinetics of cleavage as well as on cleavage site selection. The discriminator base in precurso rs to tRNA(Tyr)Su3 and tRNA(Phe) was changed from A to C and cleavage kinetics were studied by wild-type M1 RNA and a mutant M1 RNA carrying the compensatory substitution of a U to a G at position 294 in M1 RNA . Our data suggest that the discriminator base interacts with the resi due at position 294 in M1 RNA. Although product release is a rate-limi ting step both in the absence and in the presence of this interaction, its presence results in a significant reduction in the rate of produc t release. In addition, we studied cleavage site selection on various tRNA(His) precursor derivatives. These precursors carry a C at the dis criminator base position. The results showed that the mutant M1 RNA ha rboring a G at position 294 miscleaved a wild-type tRNA(His) precursor and a tRNA(His) precursor carrying an inosine at the cleavage site. T he combined data suggest a functional interaction between the discrimi nator base and the well-conserved U294 in M1 RNA. This interaction is suggested to play an important role in determining the rate of product release during multiple turnover cleavage of tRNA precursors by M1 RN A as well as in cleavage site selection.