Theses and Dissertations from UMD
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New submissions to the thesis/dissertation collections are added automatically as they are received from the Graduate School. Currently, the Graduate School deposits all theses and dissertations from a given semester after the official graduation date. This means that there may be up to a 4 month delay in the appearance of a give thesis/dissertation in DRUM
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Item Analysis of Genetic Regulatory Mechanisms that Control Ethanolamine Utilization in Enterococcus faecalis(2017) Gebbie, Margo Page; Winkler, Wade C; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)In this project, we studied the genetic regulatory mechanisms that affect utilization of ethanolamine, an abundant compound in the gastrointestinal environment. In Enterococcus faecalis, the ethanolamine utilization (eut) gene cluster encodes for a two-component regulatory system (TCS), comprised of a histidine kinase, EutW, which autophosphorylates upon sensing EA, and a cognate response regulator, EutV, which dimerizes upon receiving the phosphoryl group from EutW and binds the nascent transcript to prevent premature transcription termination. This TCS is responsible for coupling sensing of ethanolamine to production of eut transcripts. However, clues from other organisms had previously suggested that adenosylcobalamin (AdoCbl) might also be an important genetic regulatory signal for the E. faecalis eut genes. Indeed, we discovered a novel trans-acting noncoding RNA (EutX) that contained an AdoCbl-responsive riboswitch. Our data demonstrated that the riboswitch promotes a shortened form of EutX when cellular AdoCbl levels are replete. In contrast, a longer form is synthesized when AdoCbl levels are depleted. We demonstrated that structural motifs contained in the longer form of EutX act to sequester the EutV protein, preventing it from promoting transcription elongation of eut transcripts. These unexpected data revealed an important new type of regulatory mechanism for riboswitch RNAs. In support of this overall genetic regulatory model, we recapitulated the full genetic circuitry in a heterologous host. Using this system, we employed extensive site-directed mutagenesis to examine the functional importance of highly conserved EutV residues. This led to the identification of a cluster of positively charged residues, which we speculated are important determinants for RNA-binding activity. Consistent with this hypothesis, mutations of these residues resulted in loss of RNA-binding activity. Furthermore, we also explored whether the eut gene cluster was affected by additional genetic regulatory mechanisms. From these efforts, we concluded that oxygen is not a genetic regulatory feature of eut genes, in contrast to previously published speculation. However, we did find that it is likely to be repressed under conditions of high glucose. Therefore, these aggregate studies revealed new mechanisms of post-initiation genetic regulation, and showed how E. faecalis specifically controls expression of ethanolamine catabolism genes.Item NEW CHEMICAL TOOLS TO INVESTIGATE RNA FUNCTIONS(2013) Luo, Yiling; Dayie, Theodore K; Sintim, Herman O; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Ribonucleic acid (RNA), as one of the essential macromolecules of life, plays an active role in gene regulation, catalysis, and signaling because of its ability to adopt complex 3D structures that can exist in multiple conformations. Until now, RNA preparation methods devised by most investigators utilized partially denatured the RNA. The mis-folding caused by denaturing - renaturing can seriously affect RNA structure and functional activity. To test this hypothesis, in PART I of this dissertation, we presented a simple strategy using `click' chemistry to couple biotin to a `caged' photocleavable guanosine monophosphate to synthesize native RNAs that are properly folded. We demonstrated that RNA ribozymes, ranging in size from 27 to 527 nt, prepared by our non-denaturing method form a homogenous population with superior catalytic activity than those prepared by traditional refolding methods. Having developed a method for in vitro RNA synthesis, we studied the riboswitch family of RNAs that require remolding their structure for function in PART II. C-di-GMP riboswitch, as the only currently known secondary messenger riboswitch, senses c-di-GMP using its aptamer domain to modulate the expression of genes which affects biofilm formation and virulence factors production in bacteria. To specifically target pathogenic bacteria in polymicrobial systems that control the RNA-mediated c-di-GMP signaling pathway through riboswitch regulation, different c-di-GMP analogs have been used as chemical tools to investigate the structure-activity relationship (SAR) of c-di-GMP binding to different c-di-GMP riboswitches. We demonstrated that different 2'-position modified c-di-GMP analogs could differentiate between two different classes of c-di-GMP riboswitches and even bind to one particular riboswitch in class I with different affinities. Specifically, Clostridium tetani (Ct-E88) RNA in class I c-di-GMP riboswitch was used for (structural dynamic) study to understand how ligand binding drives the conformational change to regulate the downstream genes within the expression platform. Our preliminary data obtained via different biochemical and biophysical tools - such as selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE), small-angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR) spectroscopy - demonstrated that Mg2+ ions accelerate ligand recognition by pre-organizing the RNA, and then rapid ligand binding folds the RNA into a compact structure for likely downstream gene regulation.