INTERACTION OF THE 3'-END OF TRANSFER-RNA WITH RIBONUCLEASE-P RNA

Ribonuclease P, which contains a catalytic RNA subunit, cleaves 5' pre cursor-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 syn thesized by run-off transcription, tested as substrates for cleavage b y RNase P RNA and used in photoaffinity crosslinking experiments to ex amine contact sites in the ribozyme. In order to generalize the result s, analyses were carried out using three different bacterial RNase P R NAs, from Escherichia coil, Bacillus subtilis and Thermotoga maritima. At optimal (k(cat)/K-m) ionic strength (1 M NH4+/25 mM Mg2+), K-m inc reases incrementally 3- to 10-fold upon stepwise removal of each nucle otide from the 3'-end. At high ionic strength (2 M NH4+/50 mM Mg2+), w hich suppresses conformational effects, removal of the 3'-terminal A h ad 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-Watso n-Crick interaction, possibly irregular triple-strand formation with s ome component of the RNase P RNA. In agreement with previous studies, we find that the RNase P holoenzyme in vitro does not discriminate bet ween tRNAs containing or lacking CCA. The structural elements of the t hree 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 co njugated to the three RNase P RNAs by irradiation and the sites of cro sslinks 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 crossl inking results orient the CCA of tRNA with respect to that region of t he RNase P RNA.