Transcription factors (TFs) bind to specific DNA sequences near target genes to precisely coordinate their regulation. Despite the central role of transcription factors in development and homeostasis, the mechanisms by which TFs have evolved to bind and regulate distinct DNA sequences are poorly understood. This dissertation details the highly collaborative work to determine the genetic, biochemical and biophysical mechanisms by which distinct DNA-binding specificities evolved in the steroid receptor (SR) family of transcription factors. Using ancestral protein reconstruction, we resurrected and functionally characterized the historical transition in DNA-binding specificity between ancient SR proteins. We found that DNA-binding specificity evolved by changes in the energetic components of binding; interactions at the protein-DNA interface were weakened while inter-protein cooperativity was greatly improved. We identified a group of fourteen historical substitutions that were sufficient to recapitulate the derived protein's binding function. Three of these substitutions, which we defined as function-switching, were sufficient to change DNA specificity; however, their introduction greatly decreased binding affinity and was deleterious for protein function. A group of eleven permissive substitutions, which had no effect on DNA specificity, allowed for the protein to tolerate the deleterious effects of the function-switching substitutions. They non-specifically increased binding affinity by improving interactions at the protein-DNA interface and increasing inter-protein cooperativity. We then dissected the functional role of individual substitutions in both the function-switching and permissive groups. We first determined the binding affinity of all possible combinations of function-switching substitutions for a library of DNA sequences. This allowed for us to functionally characterize the sequence space that separated the ancestral and derived DNA-binding specificities as well as identify the genetic determinants for DNA specificity. Lastly, we dissected the effects of the permissive substitutions on the energetics of DNA binding to determine the mechanisms by which they exerted their permissive effect. Together, this work provides insight into the molecular determinants of DNA specificity and identifies the molecular mechanisms by which these interactions changed during the evolution of novel specificity in an important transcription factor family. This dissertation includes previously published and unpublished co-authored material. / 2016-01-14

This thesis explores the limits of homology-based inference of protein function and evolution, where overall similarity between sequences can be a poor indicator of functional similarity or evolutionary relationships. Each case presented has undergone different patterns of evolutionary change due to differing selective pressures. Surface adaptations and regulatory (e.g., gene expression) divergence are examined as molecular determinants of novel functions whose patterns are easily missed by assessments of overall sequence similarity. Following this, internal repeats and mosaic sequences are investigated as cases in which key evolutionary events involving fragments of protein sequences are masked by overall comparison. Lastly, virulence factors, which cannot be unified based on sequence, are predicted by analysis of elevated host-mimicry patterns in pathogenic versus non-pathogenic bacterial genomes. These patterns have resulted from unique co-evolutionary pressures that apply to bacterial pathogens, but may be lacking in their close relatives. A recurring theme in the proteins/genes/genomes analyzed is an involvement in microbial pathogenesis or pathogen-defense. Due to the ongoing "evolutionary arms race" between hosts and pathogens, virulence and defense proteins have undergone—and will likely continue to generate—evolutionary novelties. Thus, they demonstrate the necessity to look beyond overall sequence comparison, and assess multiple dimensions of functional innovation in proteins.

This thesis explores the limits of homology-based inference of protein function and evolution, where overall similarity between sequences can be a poor indicator of functional similarity or evolutionary relationships. Each case presented has undergone different patterns of evolutionary change due to differing selective pressures. Surface adaptations and regulatory (e.g., gene expression) divergence are examined as molecular determinants of novel functions whose patterns are easily missed by assessments of overall sequence similarity. Following this, internal repeats and mosaic sequences are investigated as cases in which key evolutionary events involving fragments of protein sequences are masked by overall comparison. Lastly, virulence factors, which cannot be unified based on sequence, are predicted by analysis of elevated host-mimicry patterns in pathogenic versus non-pathogenic bacterial genomes. These patterns have resulted from unique co-evolutionary pressures that apply to bacterial pathogens, but may be lacking in their close relatives. A recurring theme in the proteins/genes/genomes analyzed is an involvement in microbial pathogenesis or pathogen-defense. Due to the ongoing "evolutionary arms race" between hosts and pathogens, virulence and defense proteins have undergone—and will likely continue to generate—evolutionary novelties. Thus, they demonstrate the necessity to look beyond overall sequence comparison, and assess multiple dimensions of functional innovation in proteins.

Ribosomal RNA (rRNA) molecules form highly conserved secondary and tertiary structures via rRNA-rRNA and rRNA-protein interactions that collectively comprise the macromolecule that is the ribosome. Because of their cellular universality, rRNA molecules are commonly used for phylogeny estimations spanning all divergences of life. In this dissertation, I elucidate the structure of several rRNAs by analyzing multiply aligned sequences for basepair covariation and conserved higher order structural motifs. Specifically, I predict novel structures for expansion segments D2 and D3 of the nuclear large subunit rRNA (28S) and variable regions V4-V9 of the nuclear small subunit rRNA (18S) from from 249 galerucine leaf beetles (Coleoptera: Chrysomelidae). I describe a novel means for characterizing regions of alignment ambiguity that improves methods for retaining phylogenetic information without violating nucleotide positional homology. In the program PHASE, I explore a variety of RNA maximum likelihood models using the 28S rRNA dataset and discuss the utitilty of these models in light of their performance under Bayesian analysis. I conclude that seven-state models are likely the best models to use for phylogenetic estimation, although I cannot determine with confidence which of the two seven-state models (7A or 7D) is better. Evaluation of the unpaired sites within both rRNAs in Modeltest provided a similar model of evolution for these non-pairing regions (TrN+ I+G). In addition, a sequenced region of the mitochondrial cytochrome oxidase I gene (COI) from the galerucines was evaluated in Modeltest, with each codon position modeled separately (GTR+I+G for positions 1 and 2, GTR+G for position 3). The combined galerucine dataset (28S+18S rRNA helices, 28S+18S rRNA unpaired sites, COI 1st, 2nd and 3rd positions) provided for two mixedmodel Bayesian analysis of five discretely-modeled partitions (using 7A and 7D). The results of these analyses are compared with those obtained from equally weighted parsimony to provide a robust phylogenetic estimate of the Galerucinae and related leaf beetle taxa. Finally, the odd characteristics of strepsipteran 18S rRNA are evaluated through comparison of 12 strepsipterans with 163 structurally-aligned arthropod sequences. Among other interesting results, I identify errors in previously published strepsipteran sequences and predict structures not previously known from metazoan rRNA.

Thesis (M.A.)--York University, 2007. Graduate Programme in Biology. / Typescript. Includes bibliographical references (leaves 74-77). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:MR29562

Thesis (Ph. D.)--University of Texas at Austin, 2003. / Vita. Includes bibliographical references. Available also from UMI Company.

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For many species, vision contributes to a number of fitness-related tasks, including mating and the detection of prey and predators. Selection on the visual system should therefore be strong, especially when ecological or genomic changes open new avenues for evolutionary changes. Visual system proteins are thus attractive systems for molecular evolutionary analyses. This thesis presents a collection of evolutionary studies on two gene families, opsins and crystallins. Opsin proteins determine the wavelengths of light detected by the retina, while crystallin proteins contribute to lens transparency and refractory power. My studies focus on teleost fishes, because teleost visual ecology is exceptionally diverse and because gene duplication is common in this group. In Chapter One, I outline the relevance of protein variation to organismal evolution and describe the analytical methods employed throughout this thesis. Chapter Two considers the long-wavelength sensitive (LWS) opsins of the guppy (Poecilia reticulata). The guppy is shown to possess multiple LWS opsins that have accumulated differences at functionally important amino acid sites since duplicating. Chapter Three focuses on the guppy’s main predator, the pike cichlid Crenicichla frenata, which is shown to have a greater capacity for short-wavelength vision than previously believed. However, this cichlid possesses three fewer opsins than closely-related African cichlids, a difference partly due to duplication of a green-sensitive (RH2) opsin in African cichlids. In Chapter Four, this RH2 duplication event is studied in greater depth; variation in selective constraint is documented following gene duplication and between species from different lakes. Some of the analytical methods employed in Chapter Four were newly developed, as detailed in Chapter Five, where a test for functional divergence among clades is evaluated and then improved upon through the presentation of a new null model that better accommodates among-site variation in selection. In Chapter Six, phylogenetic relationships within the βγ lens crystallin superfamily are clarified, and the functionally distinct γN family is shown to have evolved conservatively compared to other crystallin families. The thesis concludes with suggestions for future directions for evolutionary research on opsins and crystallins, and summarizes recent work that has built on these studies.

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