Thesis (Ph. D.)--Florida State University, 2005. / Advisor: Dr. Hong Li, Florida State University, College of Arts and Sciences, Dept. of Chemistry and Biochemistry. Title and description from dissertation home page (viewed Sept. 19, 2005). Document formatted into pages; contains xiii, 93 pages. Includes bibliographical references.

Thesis (Ph.D.)--University of Missouri-Columbia, 2005. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file viewed on (January 25, 2007) Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Texas at Austin, 2008. / Vita. Includes bibliographical references.

Six ribosomal protein genes from the sulfur dependent extreme thermophilic archaebacterium Sulfolobus solfataricus were cloned and sequenced. Four of these genes code for proteins that are equivalent to ribosomal proteins L11, L1, L10 and L12 in Escherichia coli. The other two genes code for proteins that have no equivalent in the eubacteria. The product of one of these genes was found to be equivalent to ribosomal proteins L46 from yeast (Leer et al. 1985a) and L39 from rat liver (Lin et al. 1984), while the product of the other gene shows no sequence similarity to any of the ribosomal proteins present in the data base. In Sulfolobus, the genes that code for ribosomal proteins L11, L1, L10 and L12 are organized in the same order as in Escherichia coli, that is 5' L11, L1, L10, L12 3'. The major transcript from this gene cluster was found to be a 2.5 Kb mRNA that contains the four genes. A less abundant transcript containing only the L10 and L12 gene was also detected. Upstream of the transcription initiation sites, sequences that match the consensus sequence for archaebacterial promoters (TTTAT/AA) were found. Transcription termination sites were located within or after pyrimidine rich regions. Three of the ribosomal protein genes start with unusual initiation codons, GTG in the case of the L1 and L10 genes and TTG in the case of the L11 gene. Putative Shine Dalgarno sequences, complementary to the 3' end of Sulfolobus 16S rRNA, were detected in the region surrounding the initiation codon. In some cases (L1 and L10 genes), the initiation codon was found to be part of this sequence. Sequence comparison of the ribosomal proteins from Sulfolobus with those from other organisms, revealed that the Sulfolobus sequences are closer to those from other archaebacteria, thus supporting the existence of the archaebacterial kingdom. Comparison of the sequences of the L10 and L12 proteins from the three kingdoms revealed that the archaebacterial sequences are closer to the eukaryotes. / Graduate

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Viruses require a host cell to replicate and proliferate; upon infection they appropriate host resources and molecular machines. Specifically, viruses use ribosomes of the host to translate the information in their genome. Some viruses with single-stranded RNA genomes contain highly structured non-coding regions of RNA called internal ribosome entry sites (IRESs) which are used to hijack the host’s ribosomes through a non-canonical cap-independent initiation pathway. Canonical translation initiation is a highly complex and regulated process: at least a dozen translation factors are necessary, and it is the rate-limiting step in eukaryotic translation. Viruses containing an IRES forgo canonical eukaryotic translation initiation factors and bypass some steps of canonical translation initiation by mimicking part of the host’s initiation machinery. The simplest among these IRESs are found in the intergenic region (IGR) of viruses in the family Dicistroviridae. These type IV IRESs from dicistroviruses have been structurally characterized in great detail in using the cricket paralysis virus (CrPV) and Israeli Acute Paralysis Virus (IAPV). To better understand how structure affects the function of these type IV IRESs, using single-particle cryo-electron microscopy (cryo-EM), we have characterized a recently discovered IRES found in the IGR of the genome of Nedicistrovirus (NediV). Four complexes that represent each step in the alternative translation initiation mechanism were prepared and analyzed to solve the 3D structure and characterize the mechanism by which the NediV-IRES captures host ribosomes. With this, we were able to understand how the shorter stem-loop V (SL-V) of NediV-IRES impacts the well-characterized interaction of SL-V with eukaryotic small subunit ribosomal protein 25 (eS25) (Landry et al., 2009), which is important for the IRES:40S complex formation. This shortened stem-loop has been shown to fold in a way that does not support stable binding to the small ribosomal subunit (40S) and subsequent recruitment of the large ribosomal subunit (60S). NediV-IRES, rather, relies on direct recruitment of the 80S ribosome, which has been seen more commonly at low concentrations of Mg²⁺ for CrPV-IRES (Petrov et al., 2016). Solved structures also suggest that upon loading, NediV-IRES skips the first eEF2-dependent pseudo-translocation step necessary to bind to the ribosomal P site without the need of eEF2. Because of their simplicity, these type IV IRESs represent a robust potential tool for cell-free and vector-driven translation. Due to these structural and mechanistic differences observed, we propose that NediV-IRES, along with the NediV-like Antarctic picorna-like virus 1 (APLV-1)-IRES (Lu, 2019), represents a novel type IV IRES subclass.

The ability to sense and respond to environmental stimuli is essential for the survival of all living things. As a result, nature has evolved an uncountable number of ways to detect environmental signals. At the cellular level, G protein-coupled receptors (GPCRs) are used by eukaryotes, including fungi and humans, to convert extracellular molecular binding events into intracellular responses. Recently, synthetic biologists have shown that biological sensing systems can be repurposed to suit human needs, developing tools such as diagnostic devices and drug screening platforms. In this thesis, I present work exploring the potential of fungal GPCRs to be used as sensing elements in yeast-based biosensors. Chapter 1 gives background information related to synthetic biology, biosensors, and yeast signaling pathways. Chapter 2 describes the development of the baker's yeast Saccharomyces cerevisiae into a diagnostic device for detection of fungal pathogens, using fungal GPCRs. In Chapter 3 I demonstrate that the substrate specificity of fungal GPCRs can be altered using directed evolution. Chapter 4 describes experiments further probing the native binding abilities of fungal GPCRs, specifically examining protein ligands. Finally, in Chapter 5 we move beyond fungal GPCRs and engineer yeast to detect other stimuli, in the context of an engineered living material.

Microtubule motor proteins play fundamental roles in transporting a broad range of cellular cargoes in most eukaryotic cells. While there are over 40 kinesins helping to accommodate these diverse cellular demands, there is only one major form of cytoplasmic dynein (dynein, hereafter) carrying out almost all aspects of microtubule (MT) minus end-directed cargo transport. Dynein achieves this versatility by utilizing a wide range of adaptor proteins. Bicaudal D2 (BicD2) is a dynein adaptor protein responsible for linking cytoplasmic dynein to multiple forms of subcellular cargo. These include Rab6A, which contributes to Golgi function (Grigoriev et al., 2007; Matanis et al., 2002); the nucleoporin RanBP2 (Splinter et al., 2010); and the nucleus-cytoplasmic linker LINC complex protein Nesprin-2 (Goncalves et al., 2020). The latter two proteins were found to play important specific roles in the developing brain, respectively in the oscillatory interkinetic nuclear migration (INM) behavior characteristic of Radial Glial Progenitor (RGP) cells and in the directed nuclear migration in postmitotic neurons traveling to the expanding cortical plate. The BICD2 gene was implicated in the autosomal dominant forms of neuromuscular diseases such as Spinal Muscular Atrophy with Lower Extremity Dominance 2 (SMA-LED2) (Neveling et al., 2013; Oates et al., 2013; Peeters et al., 2013; Synofzik et al., 2014) and, more recently in at least three developmental brain pathologies: polymicrogyria (Ravenscroft et al., 2016), cerebellar hypoplasia (Fiorillo et al., 2016), and Lissencephaly (Tsai et al., 2020) expanding the clinical spectrum from neuromuscular only to potentially the entire central nervous system. It is largely unknown how these diverse clinical presentations have any relations to the BicD2 mutational sites. To investigate the genotype-phenotype relationship, it is inevitable to study each point mutation molecularly and characterize the mutational effects in in vivo setting. To investigate the mutational effects of BicD2, I used five different BicD2 fragments and nine missense mutants and characterized their behavior using biochemical and cellular approaches. Four of the missense mutations were further tested in the rat embryonic brain system, in which our lab has previously elucidated BicD2's roles during neurogenesis and post-mitotic neuronal migration. The chapters are organized by mutational effects on the BicD2-cargo interactions (Chapter 2) and the BicD2-dynein interactions (Chapter 3). In Chapter 4, I include supplemental materials to Chapters 2 and 3. In Chapter 5, I summarize, discuss, and provide my perspectives on all the mutational phenotypes found in the previous chapters. Lastly, all experimental procedures and reagents are described in Chapter 6. Here, I describe the first identification of novel gain-of-function (GoF) defects in BicD2- nuclear cargo interaction associated with two pathogenic mutations, R690C and E770G (Chapter 2). Furthermore, I characterize the GoF defects in the embryonic rat brain by in utero electroporation. The cell-type specific expression of R690C or E770G constructs revealed mutation-specific impairment of nuclear migrations in the developing cerebral cortex. In addition, I found GoF defects in BicD2-dynein interaction associated with three pathogenic mutations, L679R, R690C, and T699, surprisingly, in the C-terminal cargo binding domain of BicD2 (Chapter 3). I demonstrate that these missense mutations cause a defect in BicD2 autoinhibition control, which in turn results in abnormally enhanced dynein association. I provide evidence for hyper-activation of BicD2 for dynein binding contributes to Golgi fragmentation, which has been associated with many neuromuscular diseases (Martinez-Menarguez et al., 2019), including SMA-LED2 (Martinez-Carrera and Wirth, 2015). The entirety of this work describes molecular defects in 9 representative BicD2 mutations in vitro and demonstrates the mutational effects in vivo. I propose that the differential mutational effects associated with the BicD2 mutations might contribute to the broad spectrum of clinical phenotypes seen in patients with BicD2 diseases.

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