Distinct modes of mature and precursor tRNA binding to Escherichia coli RNase P RNA revealed by NAIM analyses

We have analyzed by nucleotide analog interference mapping (NAIM) pools of precursor or mature tRNA molecules, carrying a low level of R-p-RMP alphaS (R = A, G, I) or R-p-c7-deaza-RMP alphaS (R = A, G) modifications, to ident ify functional groups that contribute to the specific interaction with and processing efficiency by Escherichia coli RNase P RNA. The majority of inte rferences were found in the acceptor stem, T arm, and D arm, including the strongest effects observed at positions G19, G53, A58, and G71. In some cas es (interferences at G5, G18, and G71), the affected functional groups are candidates far direct contacts with RNase P RNA. Several modifications disr upt intramolecular tertiary contacts known to stabilize the authentic tRNA fold, Such indirect interference effects were informative as well, because they allowed us to compare the structural constraints required for ptRNA pr ocessing versus product binding. Our ptRNA processing and mature tRNA bindi ng NAIM analyses revealed overlapping but nonidentical patterns of interfer ence effects, suggesting that substrate binding and cleavage involves bindi ng modes or conformational states distinct from the binding mode of mature tRNA, the product of the reaction.