Ribonuclease P, which contains a catalytic RNA subunit, cleaves 5' precursor-specific sequences from pre-tRNAs. It was previously shown that the RNase P RNA optimally cleaves substrates which contain the mature, 3'-terminal CCA of tRNA. In order to determine the contributions of those individual 3'-terminal nucleotides to the interaction, pre-tRNAs that have CCA, only CC or C or are without CCA at the 3'-end were synthesized by run-off transcription, tested as substrates for cleavage by RNase P RNA and used in photoaffinity crosslinking experiments to examine contact sites in the ribozyme. In order to generalize the results, analyses were carried out using three different bacterial RNase P RNAs, from Escherichia coli, Bacillus subtilis and Thermotoga maritima. At optimal (k(cat)/K-m) ionic strength (1 M NH4+/25 mM Mg2+), K-m increases incrementally 3- to 10-fold upon stepwise removal of each nucleotide from the 3'-end. At high ionic strength (2 M NH4+/50 mM Mg2+), which suppresses conformational effects, removal of the 3'-terminal A had little effect on K-m, indicating that it is not a specific contact. Analysis of the deletion and substitution mutants indicated that the C residues act specially; their contribution to binding energy at high ionic strength (similar to 1 kcal/mol) is consistent with a non-Watson-Crick interaction, possibly irregular triple-strand formation with some component of the RNase P RNA. In agreement with previous studies, we find that the RNase P holoenzyme in vitro does not discriminate between tRNAs containing or lacking CCA. The structural elements of the three RNase P RNAs in proximity to the 9'-end of tRNA were examined by photoaffinity crosslinking. Photoagent-labeled tRNAs with 3'-terminal CCA, only CC or C, or lacking all these nucleotides were covalently conjugated to the three RNase P RNAs by irradiation and the sites of crosslinks were mapped by primer extension. The main crosslink sites are located in a highly conserved loop (probably an irregular helix) that is part of the core of the RNase P RNA secondary structure. The crosslinking results orient the CCA of tRNA with respect to that region of the RNase P RNA.
