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Higher order folding and domain analysis of the ribozyme from Bacillus subtilis ribonuclease P
1Department of Biochemistry and Molecular Biology, University of Chicago, Illinois 60637.
Abstract:
Folding of the ribozyme from Bacillus subtilis ribonuclease P (denoted P RNA) has been examined by Fe(II)-EDTA protection and bimolecular association. Fe(II)-EDTA results show that, in the presence of Mg2+, P RNA is folded into a core structure which includes most of the phylogenetically conserved nucleotides. Folding is cooperative and is complete at 5-6 mM Mg2+ with the [Mg2+]1/2 at 2-3 mM. The Hill constant indicates that this folding transition requires binding of at least three additional Mg2+ ions. Two RNA molecules consisting of nucleotides 62-239 [p(62-239)] and 240-401 + 1-61 [p(240-61)] of the B. subtilis P RNA have been constructed. These RNAs can in principle form the catalytically active structure primarily, if not solely, through tertiary interactions. Although either molecule by itself is inactive, the bimolecular complex is as active as the circularly permuted P RNAs from which it is derived. The binding constant of the complex can be as low as 0.1 microM and is strongly dependent on Mg2+ and K+ concentrations. Association of these molecules also induces a Mg2+ dependent cleavage at nucleotide 103 in p(62-239). p(62-239) gives a Fe(II)-EDTA protection pattern very similar to the wild-type P RNA at identical Mg2+ concentrations. However, Fe(II)-EDTA protection in p(240-61) is completely lost, even though it contains many nucleotides that are conserved among all P RNAs. These results suggest that, like other RNAs, P RNA contains domains that can fold in the absence of the rest of the molecule. The implications of these results are discussed in the context of the P RNA structure and catalysis.
Insights
Bacillus subtilis ribonuclease P RNA folding involves a core structure dependent on magnesium ions. Subunits can fold independently, suggesting domain-based structural organization crucial for ribozyme activity.
Area of Science:
- Biochemistry
- Molecular Biology
- RNA Structure and Function
Background:
- Ribonuclease P (P RNA) is a ribozyme essential for tRNA maturation.
- Understanding P RNA folding is key to elucidating its catalytic mechanism.
- Previous studies suggest magnesium ions play a critical role in P RNA structure.
Purpose of the Study:
- To investigate the folding pathways and structural organization of Bacillus subtilis P RNA.
- To determine the role of magnesium ions in P RNA folding.
- To explore the potential for independent domain folding within P RNA.
Main Methods:
- Fe(II)-EDTA protection assays to map RNA-protein interactions and structural integrity.
- Bimolecular association studies using truncated RNA fragments to assess domain interactions.
- Analysis of catalytic activity and binding constants of RNA complexes.
Main Results:
- Magnesium ions (Mg2+) induce cooperative folding of P RNA into a core structure containing conserved nucleotides, with optimal folding at 5-6 mM Mg2+.
- Two distinct RNA fragments, p(62-239) and p(240-61), can associate to form an active ribozyme complex, indicating tertiary interactions drive folding.
- The p(62-239) fragment retains structural features similar to wild-type P RNA, while p(240-61) shows lost protection, suggesting domain-specific folding.
Conclusions:
- P RNA folding is a Mg2+-dependent process that establishes a stable core structure.
- The P RNA molecule appears to contain independently folding domains, contributing to its overall structure and function.
- These findings provide insights into the structural basis of P RNA catalysis and its potential for modular assembly.