We have detected by nucleotide analog interference mapping
(NAIM) purine N7 functional groups in Escherichia coli
RNase P RNA that are important for tRNA binding under moderate salt
conditions (0.1 M Mg2+, 0.1 M NH4+).
The majority of identified positions represent highly or
universally conserved nucleotides. Our assay system allowed
us, for the first time, to identify c7-deaza interference
effects at two G residues (G292, G306). Several c7-deazaadenine
interference effects (A62, A65, A136, A249, A334, A351)
have also been identified in other studies performed at
very different salt concentrations, either selecting for
substrate binding in the presence of 0.025 M Ca2+
and 1 M NH4+ or self-cleavage of
a ptRNA–RNase P RNA conjugate in the presence of
3 M NH4+ or Na+. This
indicates that these N7 functional groups play a key role
in the structural organization of ribozyme–substrate
and –product complexes. We further observed that
a c7-deaza modification at A76 of tRNA interferes
with tRNA binding to and ptRNA processing by E. coli
RNase P RNA. This finding combined with the strong c7-deaza
interference at G292 of RNase P RNA supports a model in
which substrate and product binding to E. coli
RNase P RNA involves the formation of intermolecular base
triples (A258-G292-C75 and G291-G259-A76).