title: 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 (approximately 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 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.
authors.affiliation: Department of Chemistry, Indiana University, Bloomington 47405.
authors.forename: B K
authors.initials: BK
authors.lastname: Oh
authors.forename: N R
authors.initials: NR
authors.lastname: Pace
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D003432:Cross-Linking Reagents; D004722:Endoribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins; D006089:Gram-Negative Anaerobic Bacteria / Q000201:enzymology / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D010777:Photochemistry; D012313:RNA; D012322:RNA Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012343:RNA, Transfer / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D043262:Ribonuclease P; D013329:Structure-Activity Relationship
chemical_list: D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; D012322:RNA Precursors; C034477:RNA primers; D016337:RNA, Catalytic; D012313:RNA; D012343:RNA, Transfer; D004722:Endoribonucleases; D043262:Ribonuclease P; C470865:ribonuclease P, E coli