Browsing by Subject "Gene expression regulation"
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Item Novel methods for exploration and engineering of regulatory ncRNA in bacteria(2016-12-09) Amador, Paul; Contreras, Lydia M.The need for sustainable resources has spurred the establishment of microorganisms as platforms of chemical production. These “microbial factories” are engineered to maximize production of valuable chemicals. Essential to the engineering of these production strains, is the ability to control and regulate gene expression. The engineering of native and synthetic pathways of production relies on the ability to control the levels of intermediate and end products, as well as the mechanisms that impose control and induce gene expression. Traditionally, this involves the targeted deletion and overexpression of specific genes of interest within the genome. Of late, however, there has been interest in exploring the regulatory capacity of RNA for biotechnology. Discovery of regulatory elements, such as riboswitches and sponge RNAs, has advanced the capacity and utility of RNA for bioengineering. In my work, I have developed an in vivo screen for detecting RNA elements that are responsive to stress in the radiation-resistant bacteria, Deinococcus radiodurans. Investigations of the response and regulatory capacity of ncRNA, specifically 5’ UTRs, are especially valuable in this organism as radiation induces stress similar to that of oxidation and aging. Notably, this work has yielded the discovery of an mRNA-based regulon, previously thought to act only at the promoter level to regulate multiple genes associated with radiation and desiccation response. The expanded RNA-based model I present for this regulon serves to validate the advantages of RNA-level regulation for rapid and robust responses that make such RNA regulatory elements valuable for biotechnological applications. Moreover, my research into RNA regulation involved the characterization and engineering of the regulatory ncRNA, csrB, that, in concert with the regulatory protein CsrA, controls the expression of hundreds of genes throughout gamma proteobacteria. Specifically, I utilized an in vivo assay to generate an accessibility profile for csrB (for which little structural information exists in E.coli) to determine sites that are likely binding the regulatory protein, CsrA. This accessibility profile informed the rational engineering of csrB to alter affinity for binding CsrA dimers and ultimately “tune” the regulatory capacity of the Csr global regulatory system for producing complex phenotypes desirable for “microbial factories”.Item Prevalent and differential herpesviral gene regulation mediated by 3'-untranslated regions(2012-08) McClure, Lydia Virginia; Sullivan, Christopher S.Herpesviral infections are currently incurable and are associated with severe human diseases, such as cancer. Kaposi’s Sarcoma-associated Herpesvirus (KSHV), like all herpesviruses, undergoes a long-term, latent infection where few viral products are made as a mechanism to evade the host immune system. Recently, the KSHV latent genome was shown to have bivalent histone marks thought to keep the virus poised for replication. However, it is unclear how the virus prevents spurious leaky transcription from this primed state. The 3' untranslated region (3'-UTR) of transcripts is a common site of gene expression regulation, however less than half of the KSHV 3'-UTRs have been mapped and few studies have interrogated their role during infection. The work presented here is the first large-scale map and analysis of the KSHV 3'-UTRs. Four methods were used to identify the 3'-UTRs expressed by the ~85 KSHV genes, including prediction algorithms, 3'-RACE, DNA tiling array, and next generation deep sequencing analysis. The role of each KSHV 3'-UTR in gene expression was then examined using luciferase reporter assays and showed a surprising prevalence of negative regulation conveyed during latent infection. Sequential deletions across numerous 3'-UTRs indicated RNA structure is likely involved in this regulation. In addition, several KSHV 3'-UTRs conveyed an increase in translation during lytic infection through enhanced recognition by the cap-dependent translation initiation machinery activated via the MNK1 kinase. A second mechanism of KSHV gene regulation was identified through motifs encoded in the K7 3'-UTR. This work indicated that a previously characterized RNA element and a novel putative hairpin are both partially responsible for negative regulation conveyed by the K7 3'-UTR. We hypothesize that these structural motifs control expression of the K7 transcript by altering its sub-cellular location and/or via RNA stability. This work represents a broad 3'-UTR study that mapped the KSHV 3'-UTRs and is the first large-scale functional analysis of 3'-UTRs from a large genome virus. We have implicated post-transcriptional mechanisms, along with known transcriptional regulation, in viral evasion of the immune response during latency and the escape of viral-mediated host shutoff. These results identify new potential targets for therapeutic intervention of KSHV-associated disease.