Mutational analysis of the joining regions flanking helix P18 in E. coli RNase P RNA.

We have studied variants of Escherichia coli RNase P RNA with base exchanges in the joining regions flanking helix P18, which form part of the ribozyme core structure. Mutant RNase P RNAs were analyzed for: (1) specific tRNA binding by gel retardation; (2) catalytic performance in single turnover reactions; (3) structural perturbations utilizing Pb2+ -induced hydrolysis; and (4) in vivo function by complementation analysis in E. coli RNase P mutant strains. Our in vitro experiments revealed that the base moieties of nucleotides (nt) 303 and 331 to 333 neither significantly contribute to tRNA binding or structural stabilization of RNase P RNA nor to active site chemistry. Single base exchanges at nt 300, 301, and 330 reduced tRNA binding, while having little effect on the catalytic rate, which demonstrates that these nucleotides are involved in forming the high affinity (pre-)tRNA binding site. In contrast, point mutations at the strictly conserved positions nt 328, 329, 334 and 335 reduced tRNA binding affinity as well as the catalytic rate, suggesting that these mutations additionally disrupted important interactions in the catalytic center. Probing by Pb2+ revealed that particularly the mutations that affected catalytic function most strongly perturbed a more extended region (nt 248 to 335) known to be involved in tRNA binding. Under high salt conditions (> or = 0.8 M NH4+), catalytic defects of the mutant RNase P RNAs were much less pronounced, suggesting that structural perturbations leading to increased electrostatic repulsion between phosphate groups were the main cause for observed functional defects. Only mutant C334 retained a largely increased pre-steady-state K(m(pss)) under high salt conditions. We conclude that the base at position 334 is directly involved in a contact crucial to pre-tRNA binding. A complementation analysis demonstrated the important role in vivo of the joining regions flanking helix P18. None of the bases could be mutated without affecting bacterial viability.