Acquisition of novel catalytic activity by the M1 RNA ribozyme: The cost of molecular adaptation

The ribonucleoprotein RNase P is a critical component of metabolism in all known organisms. Ln Escherichia coli, RNase P processes a vast array of sub strates, including precursor-tRNAs and precursor 4.55 RNA. In order to unde rstand how such catalytic versatility is achieved and how novel catalytic a ctivity can be acquired, we evolve the M1 RNA ribozyme (the catalytic compo nent of E. coli RNase P) in vitro for cleavage of a DNA substrate. In so do ing, we probe the consequences of enhancing catalytic activity on a novel s ubstrate and investigate the cost this versatile enzyme pays for molecular adaptation. A total of 25 generations of in vitro evolution yield a populat ion showing more than a 1000-fold increase in DNA substrate cleavage effici ency (k(cat)/K-M) relative to wildtype M1 RNA. This enhancement is accompan ied by a significant reduction in the ability of evolved ribozymes to proce ss the ptRNA class of substrates but also a contrasting increase in activit y on the p4.5S RNA class of substrates. This change in the catalytic versat ility of the evolved ribozymes suggests that the acquired activity comes at the cost of substrate versatility, and indicates that E. coli RNase P cata lytic flexibility is maintained in vivo by selection for the processing of multiple substrates. M1 RNA derivatives enhance cleavage of the DNA substra te by accelerating the catalytic step (k(cat)) of DNA cleavage, although ov erall processing efficiency is offset by reduced substrate binding. The enh anced ability to cleave a DNA substrate cannot be readily traced to any of the predominant mutations found in the evolved population, and must instead be due to multiple sequence changes dispersed throughout the molecule. Thi s conclusion underscores the difficulty of correlating observed mutations w ith changes in catalytic behavior, even in simple biological catalysts for which three-dimensional models are available. (C) 1999 Academic Press.