In vitro selection of Escherichia coli RNase P: A study in molecular adaptation

The ribonucleoprotein RNase P is a critical component of metabolism in all known organisms. In E. coli, RNase P processes a vast array of substrates, including precursor-tRNAs and precursor-4.5S RNA. This dissertation focuses on understanding how RNase P catalytic versatility is achieved and how this versatility is affected by the acquisition of novel activity. To elucidate these questions, E. coli RNase P and the M1 RNA ribozyme—the catalytic component of the enzyme—are evolved in vitro for DNA cleavage. These experiments probe the consequences of inducing catalytic specialization and measure the cost of molecular adaptation to this versatile enzyme.; The first set of experiments evolves a population of M1 RNA ribozyme derivatives for DNA cleavage in the absence of the RNase P protein subunit (C5 protein). Twenty-five generations of in vitro evolution yield a population showing a 1000-fold increase in DNA cleavage efficiency (k_cat/K_M) relative to wild-type M1 RNA. This enhancement is accompanied by an inability to process a natural ptRNA substrate, indicating evolved ribozymes lose substrate versatility. Enhanced DNA cleavage cannot be readily traced to any single point mutation, resulting instead from multiple sequence changes. This conclusion underscores the difficulty of correlating observed mutations with changes in catalytic behavior, even in catalysts for which three-dimensional models are available.; The second set of experiments explores the effect of C5 protein on M1 RNA evolution. Ten generations of in vitro selection produced RNase P derivatives showing a 100-fold improvement in DNA cleavage efficiency relative to the wild-type holoenzyme. These evolved ribozymes require C5 protein to enhance DNA cleavage, indicating that the novel activity is mediated by RNA-protein interactions. Evolved holoenzymes also show a loss of substrate versatility and C5 protein is unable to mitigate the tradeoff between enhanced DNA cleavage and reduced ptRNA cleavage. This change in the catalytic versatility of evolved RNase P suggests that the catalytic flexibility displayed by this enzyme in vivo is maintained by natural selection for the processing of multiple substrates.