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While critical cellular components-such as the RNA moiety of bacterial ribonuclease P-can sometimes be replaced with a highly divergent homolog, the cellular response to such perturbations is often unexpectedly complex. RNase P is a ubiquitous and essential ribonucleoprotein involved in the processing of multiple RNA substrates, including tRNAs, small non-coding RNAs and intergenic operons. In Bacteria, RNase P RNAs have been subdivided-based on their secondary and tertiary structures-into two major groups (A and B), each with a distinct phylogenetic distribution. Despite the vast phylogenetic and structural gap that separates the two RNase P RNA classes, previous work suggested their interchangeability. Here, we explore in detail the functional and fitness consequences of replacing the endogenous Type-A Escherichia coli RNase P RNA with a Type-B homolog derived from Bacillus subtilis, and show that E. coli cells forced to survive with a chimeric RNase P as their sole source of RNase P activity exhibit extremely variable responses. The chimeric RNase P alters growth rates-used here as an indirect measure of fitness-in unpredictable ways, ranging from 3- to 20-fold reductions in maximal growth rate. The transcriptional behavior of cells harboring the chimeric RNAse P is also perturbed, affecting the levels of at least 79 different transcripts. Such transcriptional plasticity represents an important mechanism of transient adaptation which, when coupled with the emergence and eventual fixation of compensatory mutations, enables the cells to overcome the disruption of this tightly coevolving ribonucleoprotein.

Escherichia coli RNase P derivatives were evolved in vitro for DNA cleavage activity. Ribonucleoproteins sampled after ten generations of selection show a >400-fold increase in the first-order rate constant ( k cat ) on a DNA substrate, reflecting a significant improvement in the chemical cleavage step. This increase is offset by a reduction in substrate binding, as measured by K M . We trace the catalytic enhancement to two ubiquitous A → U sequence changes at positions 136 and 333 in the M1 RNA component, positions that are phylogenetically conserved in the Eubacteria. Furthermore, although the mutations are located in different folding domains of the catalytic RNA, the first in the substrate binding domain, the second near the catalytic core, their effect on catalytic activity is significantly influenced by the presence of the C5 protein. The activity of the evolved ribonucleoproteins on both pre-4.5 S RNA and on an RNA oligo substrate remain at wild-type levels. In 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, despite 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 single-stranded region, likely alters the geometry of the pre-tRNA-binding cleft and may provide a functional explanation for the observed tradeoff. These 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 repertoire of RNA.

The evolution of any given protein reflects the interplay between proximal selective forces involving the conservation of protein structure and function and more general populational factors that shape the action and efficiency of natural selection. In an attempt to address that interplay, we have analyzed patterns of amino acid replacement within a well-conserved molecule, alcohol dehydrogenase (ADH), in the Drosophilidae. A sliding window, moved along the protein sequence in order to quantify the extent of change at each amino acid position, reveals heterogeneous amounts of replacement across the molecule when all ADH sequences are analyzed simultaneously. Surprisingly, the replacement profile for ADH differs significantly in the melanogaster, mulleri, and Hawaiian subgroups, reflecting the imprint of the differing evolutionary histories of each of these assemblages on the evolution of this conservative molecule.

In order to explore the variety of possible responses available to a ribozyme population evolving a novel phenotype, five Tetrahymena thermophila group I intron ribozyme pools were evolved in parallel for cleavage of a DNA oligonucleotide. These ribozyme populations were propagated under identical conditions and characterized when they reached apparent phenotypic plateaus; the populations that reached the highest plateau showed a near 100-fold improvement in DNA cleavage activity. A detailed characterization of the evolved response in these populations reveals at least two distinct phenotypic trajectories emerging as a result of the imposed selection. Not only do these distinct solutions exhibit differential DNA cleavage activity, but they also exhibit a very different correlation with a related, but unselected, phenotype: RNA cleavage activity. In turn, each of these trajectories is underwritten by differing genotypic profiles. This study underscores the complex network of possible trajectories through sequence space available to an evolving population and uncovers the diversity of solutions that result when the process of experimental evolution is repeated multiple times in a simple, engineered system.

The Drosophila olfactory genes OS-E and OS-F are members of a family of genes that encode insect odorant-binding proteins (OBPs). OBPs are believed to transport hydrophobic odorants through the aqueous fluid within olfactory sensilla to the underlying receptor proteins. The recent discovery of a large family of olfactory receptor genes in Drosophila raises new questions about the function, diversity, regulation, and evolution of the OBP family. We have investigated the OS-E and OS-F genes in a variety of Drosophila species. These studies highlight potential regions of functional significance in the OS-E and OS-F proteins, which may include a region required for interaction with receptor proteins. Our results suggest that the two genes arose by an ancient gene duplication, and that in some lineages, one or the other gene has been lost. In D. virilis, the OS-F gene shows a different spatial pattern of expression than in D. melanogaster. One of the OS-F introns shows a striking degree of conservation between the two species, and we identify a putative regulatory sequence within this intron. Finally, a phylogenetic analysis places both OS-E and OS-F within a large family of insect OBPs and OBP-like proteins.

The ribonucleoprotein RNase P is a critical component of metabolism in all known organisms. In Escherichia coli, RNase P processes a vast array of substrates, including precursor-tRNAs and precursor 4. 5S RNA. In order to understand how such catalytic versatility is achieved and how novel catalytic activity can be acquired, we evolve the M1 RNA ribozyme (the catalytic component of E. coli RNase P) in vitro for cleavage of a DNA substrate. In so doing, we probe the consequences of enhancing catalytic activity on a novel substrate and investigate the cost this versatile enzyme pays for molecular adaptation. A total of 25 generations of in vitro evolution yield a population showing more than a 1000-fold increase in DNA substrate cleavage efficiency (kcat/KM) relative to wild-type M1 RNA. This enhancement is accompanied by a significant reduction in the ability of evolved ribozymes to process the ptRNA class of substrates but also a contrasting increase in activity on the p4.5S RNA class of substrates. This change in the catalytic versatility of the evolved ribozymes suggests that the acquired activity comes at the cost of substrate versatility, and indicates that E. coli RNase P catalytic flexibility is maintained in vivo by selection for the processing of multiple substrates. M1 RNA derivatives enhance cleavage of the DNA substrate by accelerating the catalytic step (kcat) of DNA cleavage, although overall processing efficiency is offset by reduced substrate binding. The enhanced 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. This conclusion underscores the difficulty of correlating observed mutations with changes in catalytic behavior, even in simple biological catalysts for which three-dimensional models are available.