Protein cofactor-dependent acquisition of novel catalytic activity by the RNase p ribonucleoprotein of E-coli

Escherichia coli RNase P derivatives were evolved in vitro for DNA cleavage activity. Ribonucleoproteins sampled after ten generations of selection sh ow a > 400-fold increase in the first-order rate constant (k(cat)) on a DNA substrate, reflecting a significant improvement in the chemical cleavage s tep. This increase is offset by a reduction in substrate binding, as measur ed by K-M. We trace the catalytic enhancement to two ubiquitous A --> U seq uence changes at positions 136 and 333 in the M1 RNA component, positions t hat are phylogenetically conserved in the Eubacteria. Furthermore, although the mutations are located in different folding domains of the catalytic RN A, the first in the substrate binding domain, the second near the catalytic core, their effect on catalytic activity is significantly influenced by th e presence of the C5 protein. The activity of the evolved ribonucleoprotein s on both pre-4.5 S RNA and on an RNA oligo substrate remain at wild-type l evels. Ln contrast, improved DNA cleavage activity is accompanied by a 500- fold decrease in pre-tRNA cleavage efficiency (k(cat)/K-M). The presence of the C5 component does not buffer this tradeoff in catalytic activities, de spite the in vivo role played by the C5 protein in enhancing the substrate versatility of RNase P. The change at position 136, located in the J11/12 s ingle-stranded region, likely alters the geometry of the pre-tRNA-binding c left and may provide a functional explanation for the observed tradeoff. Th ese results thus shed light both on structure/function relations in E. coli RNase P and on the crucial role of proteins in enhancing the catalytic rep ertoire of RNA. (C) 2001 Academic Press.