MUTATIONAL ANALYSIS OF THE JOINING REGIONS FLANKING HELIX P18 IN ESCHERICHIA-COLI RNASE-P RNA

We have studied variants of Escherichia coli RNase P RNA with base exc hanges in the joining regions flanking helix P18, which form part of t he ribozyme core structure. Mutant RNase P RNAs were analyzed for: (1) specific tRNA binding by gel retardation; (2) catalytic performance i n single turnover reactions; (3) structural perturbations utilizing Pb 2+-induced hydrolysis; and (4) in vivo function by complementation ana lysis in E. coli RNase P mutant strains. Our in vitro experiments reve aled that the base moieties of nucleotides (nt) 303 and 331 to 333 nei ther significantly contribute to tRNA binding or structural stabilizat ion of RNase P RNA nor to active site chemistry. Single base exchanges at nt 300, 301 and 330 reduced tRNA binding, while having little effe ct on the catalytic rate, which demonstrates that these nucleotides ar e involved in forming the high affinity (pre-)tRNA binding site. In co ntrast, point mutations at the strictly conserved positions nt 328, 32 9, 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 p articularly the mutations that affected catalytic function most strong ly perturbed a more extended region (nt 248 to 335) known to be involv ed in tRNA binding. Under high salt conditions (greater than or equal to 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 ma in 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 demons trated the important role in vivo of the joining regions flanking heli x P18. None of the bases could be mutated without affecting bacterial viability. (C) 1996 Academic Press Limited