Ribonuclease P (RNase P) RNA is converted to a Cd(2+)-ribozyme by a single Rp-phosphorothioate modification in the precursor tRNA at the RNase P cleavage site.

Abstract

To study the cleavage mechanism ofbacterial Nase P RNA, we have synthesized precursor tRNA substrates carrying a single Rp-or RNase P is an essential structure-specific endoribonuclease that generates the mature 5' ends of tRNAs. In vitro, RNA subunits of bacterial RNase P enzymes were shown to be catalytically active in the absence of the protein subunit (1). Processing of precursor tRNAs (ptRNAs) by RNase P is an essentially irreversible reaction yielding 3'-OH and 5'-phosphate termini. A solvent hydroxide is thought to act as the nucleophile in an SN2 in-line displacement mechanism (2, 3).To gain a deeper insight into the cleavage mechanism by Escherichia coli RNase P RNA, we have synthesized ptRNA substrates carrying a single Rp-or Sp-phosphorothioate modification at the RNase P cleavage site. The diastereomeric substrates were analyzed for gel-resolvable binding to RNase P RNA and were studied in single turnover experiments in the presence of different divalent metal ions, such as Mg2+, Mn2+, and Cd2+. The following results were obtained: (i) the Spdiastereomer moderately affected ptRNA ground state binding, while the Rp-diastereomer had no effect; (ii) cleavage by RNase P RNA involves direct metal ion coordination to the pro-Rp oxygen; (iii) there is no specific role for Mg2+ at the pro-Sp oxygen; and (iv) cleavage of the Rp-diastereomeric substrate has a lower cooperative dependence (nH = 1.8) upon[Cd2+] than cleavage of the unmodified substrate upon [Mg2+] (nH = 3.3). Implications for the unique RNase P RNA cleavage mechanism are discussed in the context of previously proposed mechanistic models (2-4). MATERIALS AND METHODS Separation and Identification of Diastereomeric 13-Mers.Oligoribonucleotides of identical sequence (13-mers), either unmodified or carrying a single phosphorothiote modification (5'-CCCUUUCsGCGGGA), were prepared by solid-phase synthesis essentially as described (5, 6). Oligoribonucleotides were purified by reversed-phase HPLC on an ODS C18 Beckman Ultrasphere column at 45°C (5). The material of the peak containing the two diastereomeric 13-mers was vacuumdried, and the Rp-and Sp-diastereomers were separated by a second reversed-phase run at 4°C. The assignment of configuration of the two diastereomeric 13-mers was accomplished by digestion with the stereoselective enzymes snake venom phosphodiesterase I or nuclease P1 essentially as described (6). Phosphorothioate-specific iodine hydrolysis was performed essentially as described recently (7).Assembly of the ptRNAGIY. Chemically synthesized RNA oligomers were phosphorylated at their 5' termini by T4 polynucleotide kinase; T7 transcripts were synthesized in the presence of excess 5'-GMP to obtain 5'-monophosphates (7). Modified or unmodified 13-mers, a second 11-nt RNA oligonucleotide (5'-GUAGCUCAGUC-3', obtained either by chemical RNA synthesis or T7 transcription), and the 3'-portion of the tRNA (obtained by T7 transcription, starting at G+ 18; see Fig. 1) were annealed to a bridging DNA oligonucleotide (complementary to...

Biochemical Characterization of a Lanthanide-Dependent DNAzyme with Normal and Phosphorothioate-Modified Substrates

“…the hammerhead ribozyme, 32 HDV ribozyme, 33 [10][11][12][13][14][15][16][17][18][19][20][21][22][23] DNAzyme, 37 RNase P, 38 Group II intron, 39 ) use pro-Rp for metal binding (mostly Mg 2+ ). This study also indicates that the pro-Rp oxygen is more important for metal binding in the Ce13d DNAzyme.…”

Metal ion cooperativity in ribozyme cleavage of RNA

“…The roles of Mg 2ϩ in the RNase P RNA-catalyzed reaction are to induce proper folding of the RNA, to facilitate the interaction with its RNA substrate, and to promote efficient and correct cleavage (7). Cleavage by RNase P RNA has been reported to proceed in the presence of Mn 2ϩ and Ca 2ϩ , although at reduced efficiencies (2,(8)(9)(10)(11)(12)(13)(14)(15). However, cleavage in the presence of Mn 2ϩ and Ca 2ϩ induces miscleavage (11,12,15).…”

Probing the Role of Metal Ions in RNA Catalysis: Kinetic and Thermodynamic Characterization of a Metal Ion Interaction with the 2‘-Moiety of the Guanosine Nucleophile in the Tetrahymena Group I Ribozyme

“…Numerous metal ions typically coat an RNA to neutralize the backbone charges and stabilize tertiary contacts, facilitating the folding of RNA into specific threedimensional structures (e.g., 2-19 and references cited therein). In addition to these structural roles, specific active site metal ions also directly participate in the chemical transformations catalyzed by many RNA enzymes (e.g., [20][21][22][23][24][25][26]. However, the sea of metal ions bound to RNA presents an enormous challenge to deciphering the role of individual metal ions in RNA catalysis.…”

“…Specific metal ion ligands have been identified by metal ion specificity switch experiments (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32). These experiments exploit the preference of nitrogen and sulfur ligands to interact more strongly with soft metal ions, such as Mn 2+ , than with hard metal ions, such as Mg 2+ .…”