Theses and Dissertations from UMD
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Item INVESTIGATING THE COMPETITION BETWEEN COMPONENTS OF DUAL-FUNCTION RNA(2021) Aoyama, Jordan James Masuo; Storz, Gisela T; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Non-coding RNAs (ncRNAs) and small proteins have both emerged as important regulators of gene expression. Dual-function RNAs encode a small protein and have a separate function as a regulatory RNA. Although first discovered in bacteria, dual-function RNAs have now been identified and characterized in eukaryotes as well. These RNAs allow two activities of a single gene to regulate targets at multiple levels. The work described here explores how two novel and one synthetic dual-function RNA act and how competition between the components of a dual-function RNA impacts their functions. AzuCR is a 164-nucleotide E. coli RNA that was previously shown to encode a 28 amino acid protein (AzuC). This work demonstrates that the AzuC small protein impacts glycerol metabolism, with the small protein increasing activity of GlpD, an essential enzyme in glycerol catabolism, while the RNA base pairs with and represses galE mRNA, a gene essential for galactose metabolism. The second dual-function RNA studied in this work is Spot 42, a 109-nucleotide RNA known to base pair with and repress mRNAs encoding proteins involved in the metabolism of non-preferred carbon sources. Although Spot 42 is a well-characterized base pairing small RNA (sRNA) in E. coli, this work shows it also encodes a 15-amino acid protein, SpfP. SpfP was found to bind to cAMP receptor protein (CRP) and block activation of some target genes. For both AzuCR and Spot 42, the coding sequence for the small protein overlaps the base pairing region, and we have observed that translation interferes with base pairing activity suggesting competition between the sRNA and mRNA activities. Finally, a synthetic dual-function RNA was constructed from the Escherichia coli sRNA MgrR and the mRNA for the small protein MgtS. Various versions of this hybrid molecule are used to probe how the organization of components is important for the proper functioning of a dual-function RNA. These three studies highlight the complexities of regulation by dual-function RNAs and provide insights into how these molecules coordinate two different activities to carry out regulatory roles in the cell.Item Harnessing the Potential of the Escherichia coli RpoS Phenotype via an Inducible Small RNA Regulatory Platform(2011) Carter, Karen; Bentley, William E; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Recent recognition of the pervasiveness of non-coding RNAs, in both prokaryotic and eukaryotic systems, has prompted metabolic engineers to reevaluate the role of RNAs in a traditionally protein dominated realm. More specifically, bacterial trans-encoded sRNAs have been implicated in the regulation of genes in several critical pathways from quorum sensing to stress responses. The task of responding to stressful conditions, as well as stationary phase, in a comprehensive manner falls to the Escherichia coli global stress regulator, RpoS. Genes transcribed by RpoS are involved in motility, biofilm formation and nutrient limitations. One of the challenges modulating RpoS control is its polymorphic nature. We think this can be addressed using an inducible sRNA regulatory platform. Recent studies have confirmed RpoS to be post-transcriptionally regulated by at least four sRNAs: three activators, DsrA, RprA and ArcZ, and one repressor OxyS. Each of these senses different stress conditions, allowing RpoS synthesis to increase or decrease in response to various stressors. This work investigates the potential of a genetically engineered interchangeable small RNA based gene regulation platform as a switch to affect the expression profiles and metabolic behavior of RpoS. RprA and OxyS were put under the control of an arabinose inducible promoter to test the ability to increase/decrease RpoS protein levels and subsequent changes in RpoS-dependent genes. We then assessed gene expression and phenotypic changes using RT-PCR, Western blotting, microarray and motility and biofilm assays. Positive modulation of RpoS using the pRprA platform resulted in a 2-fold decrease in motility in Top10 cells. This difference in motility improved biofilm formation levels up to 12-fold when compared to direct overexpression of RpoS protein. The positive effect of biofilm formation was further supported by the upregulation of other genes essential for biofilms. Conversely, negative modulation of RpoS using the pOxyS platform resulted in an increase in the transcription of the motility gene, flhD. Both systems were capable of positively and negatively regulating bacterial RpoS protective genes. The ability to deliberately and purposefully control RpoS protective genes, in conjunction with motility and biofilm formation, can potentially have broad impact on biotechnology applications.Item Evaluation of the transcription of small RNA SgrS and glucose transporter mRNA ptsG in E. coli B and E. coli K cultures under high glucose conditions(2009) Ng, Weng Ian; Wang, Nam Sun; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Escherichia coli is commonly used as the production system for recombinant proteins. However, acetate accumulation in fermentation affects cell growth and protein yield. Recent studies have showed that the small RNA SgrS regulates the major glucose transporter mRNA ptsG in a post–transcriptional manner when the metabolic intermediate glucose–6–phosphate is accumulated intracellularly in E. coli K. Here, comparative analysis of the transcription of SgrS and ptsG is performed between E. coli B and E. coli K cultures in both shake flasks and bioreactor. Both strains expressed SgrS when grown on the non–metabolizable glucose analog α–methyl–glucoside. However, under high glucose conditions, only E. coli B showed significant expression of SgrS. This behavior is unaffected by oxygen supply and pH control. E. coli B produced less acetate on glucose than E. coli K in the bioreactor settings. This provides evidence of a possible connection between SgrS and acetate production in aerobic fermentation of E. coli.