Other Microbiology Commons^™

Mutational Analysis Of Domain Functions In The Yeast Decapping Enzyme Dcp2, Jellisa Ewan, Ambro Van Hoof

Dissertations and Theses (Open Access)

In yeast, the major mRNA decay pathway involves deadenylation of the 3’ poly-A tail, followed by decapping of the 5’ m7Gppp cap and subsequent 5’ to 3’ degradation or 3’ to 5’ degradation. This is carried out by Pan2/Pan3 and Ccr4/Not deadenylases, the decapping enzyme Dcp2, and Xrn1 and  RNA exosome exoribonucleases, respectively. The eukaryotic mRNA decapping enzyme complex, Dcp1/Dcp2, is essential in yeast, and deletion of either gene is lethal. However, the deadenylases that act upstream and the 5’ exoribonuclease that act downstream of the decapping step are not, even though they are all highly conserved with no known …

Structure And Function Studies On Trna Splicing Endonuclease, Justin G. Underwood

Dissertations and Theses (Open Access)

A subset of eukaryotic pre-tRNAs have introns that must be spliced out to become mature molecules capable of functioning in translation. This essential processing step is performed by tRNA splicing endonuclease (TSEN), a highly conserved heterotetramer with two catalytic and two structural subunits. In addition to this conserved function, archaeal TSEN also processes pre-rRNA and yeast TSEN cleaves some mRNAs that encode mitochondrial proteins to initiate mRNA degradation. Interestingly, single amino acid mutations in TSEN cause the human Mendelian disease pontocerebellar hypoplasia (PCH) by an unknown mechanism. Recently, several groups have determined the structure of human TSEN, revealing similarities and …

Differential Progression Of Pancreatic Premalignancy In Geographically Distant Murine Transgenic Cohorts Uncovers Key Role Of Microbial Metabolites, Rian M. Howell

Dissertations and Theses (Open Access)

Cancer is a leading cause of death worldwide. Despite recent advances in diagnostic and therapeutic modalities, pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer that remains highly lethal. Efforts have been made to increase the understanding of the underlying mechanisms driving early stages of tumor development, when the cancer is most susceptible to intervention. To this end, genetically engineered mouse models (GEMMs) have been developed with the mutations most commonly seen in the patient population. Expression of these mutations in select cellular compartments has resulted in the ability to recapitulate characteristics of human pancreatic cancer such as the development of …

Functions Of The Trna Splicing Endonuclease And Other Adventures In Rna Processing, Jennifer Hurtig, Ambro Van Hoof

Dissertations and Theses (Open Access)

The tRNA splicing endonuclease (TSEN), has been studied for over three decades for its function in tRNA splicing. However, this enzyme has other functions that are just beginning to be characterized. Mutations in TSEN cause the neuronal disease pontocerebellar hypoplasia (PCH) that is characterized by atrophy of the cerebellum and pons, overall developmental failure, and usually results in death before adolescence. How mutations in TSEN cause these neuronal defects and disease is not understood. In yeast, TSEN has another essential function that is independent of tRNA splicing and is still unknown. In this thesis I strived to understand the other …

Thiol-Based Misfolding: Linking Redox Balance To Cytosolic Proteostasis, Ford Amy

Dissertations and Theses (Open Access)

The eukaryotic cytosolic proteome is vulnerable to changes in proteostatic and redox balance caused by temperature, pH, oxidants and xenobiotics. Cysteine-containing proteins are especially at risk as the thiol side chain is subject to oxidation, adduction and chelation by thiol-reactive compounds. All of these thiol-modifiers have been demonstrated to induce the heat shock response and recruit protein chaperones to sites of presumed protein aggregation in the budding yeast Saccharomyces cerevisiae. However, endogenous targets of thiol stress toxicity responsible for these outcomes are largely unknown. Furthermore, I hypothesize proteins identified as redox-active are prone to misfolding and aggregation by thiol-specific …

Hsp70-Mediated Regulation Of Hsf1 Transcriptional Activity In Saccharomyces Cerevisiae, Sara Peffer

Dissertations and Theses (Open Access)

In eukaryotic cells, protein homeostasis and cellular fitness is promoted by the transcription factor heat shock factor 1 (HSF1) during exposure to proteotoxic stress. HSF1 controls the basal and stress-induced expression of molecular chaperones and other protective targets. Dynamic regulation of HSF1 involves the major heat shock proteins Hsp70 and Hsp90. Recent advances in the understanding of this regulatory circuit in Saccharomyces cerevisiae have shown that the Hsp70 Ssa1 acts as a sensor for some proteotoxic stresses and is capable of a direct interaction with Hsf1. This work continues to explore the complex regulatory interaction between Hsf1 and Ssa1. I …

Mechanism Of Incorporation And Repair Of Uracil At Highly Transcribed Genes In Saccharomyces Cerevisiae, Norah Auma Owiti

Dissertations and Theses (Open Access)

Recombination and mutagenesis are elevated by high levels of transcription. The correlation between transcription and genome instability is largely explained by the topological and structural changes in DNA and the associated physical obstacles generated by the transcription machinery. However, such explanation does not directly account for the unique types of mutations originating from the non-canonical residues such as uracil, which are also elevated at highly transcribed regions. Apurinic/Apyrimic or Abasic (AP) sites derived from uracil excision, accumulate at a higher rate in actively transcribed regions of the genome in S. cerevisiae and are primarily repaired by base excision repair (BER) …

Functional Consequences Of Rna Exosome Complex Alteration By Conformational Changes And Cofactor Binding, Jaeil Han

Dissertations and Theses (Open Access)

The RNA exosome is an essential 3’-5 ribonuclease that processes or degrades a variety of RNA species in eukaryotes. It is composed of nine structural cores and one catalytic subunit, Rrp44. Structural studies captured two different conformations of Rrp44, Rrp44^ch (channel) and Rrp44^da (direct-access). The Rrp44^ch appears to recruit RNA substrates from the central channel formed by the core subunits, while the substrate is directly recruited to Rrp44^da bypassing the central channel. Although in vivo function of the Rrp44^ch-exosome is extensively studied, the function or even the presence of the Rrp44^da-exosome in …

Quantitative Analysis And Imaging-Based Insights Into The Characteristics And Mechanisms Of Yeast Pattern Formation, Lin Chen

Dissertations and Theses (Open Access)

Biofilm formation is a common lifestyle adapted by bacteria and fungi in response to various environmental stresses. Bacterial and fungal biofilms adhering to medical devices convey resistance to antibiotics or biocides, causing high rates of clinical infections. Microorganisms are protected from harsh environmental conditions by reduced stress penetration through the complex biofilm architecture with distinct patterns. Although the molecular regulations of surface patterning have been well characterized in bacteria, the mechanisms underlying the complex pattern formation in eukaryotic biofilms remain unclear.

This dissertation aims to investigate the salient features of robust colony expansion in yeast biofilms and the processes driving …

Development Of A Molecular Gram-Stain Assay For The Diagnosis Of Blood Stream Infections Associated With Sepsis, Douglas Bryan Litwin

Dissertations and Theses (Open Access)

Sepsis is a serious medical condition resulting from the severe dysregulation of the immune response that is generally triggered by infection. It affects more than 1.1 million Americans, has an average mortality rate of 30%, and is estimated to cost $24.3 billion annually. Currently, blood culture followed by Gram-stain analysis is the gold standard for diagnosing bacterial infections associated with sepsis. This method generates a high rate of false negative results and, in general, requires 20 to 48 hr to provide results. Both of these problems are related to the requirement that the bacterial pathogens grow under defined laboratory conditions. …

The Role Of The Arched Helicases In Exosome-Mediated Function, A. Alejandra Klauer

Dissertations and Theses (Open Access)

RNA processing and degradation are two important functions that control gene expression and promote RNA fidelity in the cell. A major ribonuclease complex, called the exosome, is involved in both of these processes. The exosome is composed of ten essential proteins with only one catalytically active subunit, called Rrp44. While the same ten essential subunits make up both the nuclear and cytoplasmic exosome, there are nuclear and cytoplasmic exosome cofactors that promote specific exosome functions in each of the cell compartments. To date, it is unclear how the exosome distinguishes between RNA substrates. We hypothesize that compartment specific cofactors may …

Multiple Posttranscriptional Regulatory Features Control Expression Of Ethanolamine Utilization Genes In Enterococcus Faecalis, Kristina A. Fox

Dissertations and Theses (Open Access)

Enterococcus faecalis is a Gram-positive bacterium that lives as a commensal organism in the mammalian gastrointestinal tract, but can behave as an opportunistic pathogen.  Our lab discovered that mutation of the eutK gene attenuates virulence of E. faecalis in the C. elegans model host.  eutK is part of the ethanolamine metabolic pathway which was previously unknown in E. faecalis.  I discovered the presence of two unique posttranscriptional regulatory features that control expression of eut locus genes.  The first feature I found is an AdoCBL riboswitch, a cis-acting RNA regulatory element that acts as a positive regulator of gene …

Stress-Induced Targeting Of Molecular Chaperones In The Yeast Saccharomyces Cerevisiae, Hugo Tapia

Dissertations and Theses (Open Access)

The eukaryotic stress response is an essential mechanism that helps protect cells from a variety of environmental stresses. Cell death can result if cells are not able to properly adapt and protect themselves against adverse stress conditions. Failure to properly deal with stress has implications in human diseases including neurodegenerative disorders and distinct cancers, emphasizing the importance of understanding the eukaryotic stress response in detail. As part of this response, expression of a battery of heat shock proteins (HSP) is induced, which act as molecular chaperones to assist in the repair or triage of unfolded proteins. The 90-kDa HSP (Hsp90) …

The Domains Of The Catalytic Subunit Of The Eukaryotic Rna Degrading Exosome, Rrp44p, Have Distinct Functions, Daneen Schaeffer

Dissertations and Theses (Open Access)

The exosome is a 3’ to 5’ exoribonuclease complex that consists of ten essential subunits. In the cytoplasm, the exosome degrades mRNA in a general mRNA turnover pathway and in several mRNA surveillance pathways. In the nucleus, the exosome processes RNA precursors to form small, stable, mature RNA species, including rRNA, snRNA, and snoRNA. In addition to processing these RNAs, the nuclear exosome is also involved in degrading aberrantly processed forms of these RNAs, and others, including mRNA.

The 3’ to 5’ exoribonuclease activity of the exosome is contributed by the RNB domain of the only catalytically active subunit, Rrp44p, …