HIGHER-ORDER FOLDING AND DOMAIN ANALYSIS OF THE RIBOZYME FROM BACILLUS-SUBTILIS RIBONUCLEASE-P

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 mu M 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.