Interaction of the 3′-end of tRNA with ribonuclease P RNA

Abstract

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 (kcat/Km) ionic strength (1 M NH4 $/25 mM Mg2$), Km 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 Km, 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 (̃1 kcal/mol) is consistent with a non- Watson - Crick interaction, possibly irregular triplestrand 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 3′-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. © 1994 Oxford University Press.