Nucleic Acids Research, 1994, Vol. 22, No. 20 4087-4094
© 1994
RNA |
Interaction of the 3'-end of tRNA with ribonuclease P RNA
1Departments of Chemistry Indiana University Bloomington, IN 47405, USA 2Departments of Biology Indiana University Bloomington, IN 47405, USA 3institute for Molecular and Cellular Biology, Indiana University Bloomington, IN 47405, USA
*To whom correspondence should be addressed
Received July 15, 1994. Revised August 25, 1994. Accepted August 25, 1994.
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 ({small tilde}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.
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