Cell Biology & Molecular Genetics Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/2750
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Item Programmed Ribosomal Frameshifting in SARS-CoV and HIV-1(2007-12-10) Neeriemer, Jessica; Dinman, Jonathan D; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Programmed ribosomal frameshifting controls the ratio of two protein products made in a variety of viruses and mammalian cells. This occurs when the ribosome is translating mRNA, pauses at secondary structure, slips back one base in the 5' direction, and continues translation in a new reading frame. A series of SARS-CoV pseudoknot mutants were generated to examine important features of frameshifting, and an antibiotic was tested for its effect on HIV and SARS-CoV frameshifting. Other mutants were made in the human CCR5 gene to determine whether frameshifting occurs. It was found that mRNA stability and unpaired adenosines influence frameshifting, and increasing concentrations of the antibiotic gentamicin increases frameshifting. Moreover, CCR5, the co-receptor for HIV, contains a working frameshifting signal. This study pinpoints several antiviral targets and important factors for HIV and SARS-CoV pathogenesis.Item Wiring the ribosome: functions of ribosomal proteins L3 and L10, and 5S rRNA(2006-09-29) Petrov, Alexey; Dinman, Jonathan D; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The ribosome is a megadalton complex that performs protein synthesis with tremendous speed and accuracy. Atomic resolution ribosome structures have been resolved within the last five years. These have provided the 3-dimensional locations of all ribosomal components, and have revealed structures of the active centers. However, the precise mechanisms of the various functions performed by the ribosome are still unknown. This work is an attempt to understand some of the functional relationships between different active centers of the ribosome (or the "wiring" of the ribosome), and mechanisms by which such communication occurs. Here we present an analysis of three ribosomal components: ribosomal proteins L3 and L10, and 5S rRNA. Studies of L3 suggest that accommodation of aminoacyl-tRNAs (aa-tRNA) may be the mechanism that induces the "active" conformation of the peptidyl transferase center. We have proposed a mechanism in which rRNA movement associated with aa-tRNA accommodation facilitates conformational changes in the peptidyl transferase center (PTC) through the formation of a network of hydrogen bond interactions. A saturation mutagenesis analysis of 5S rRNA disproves the previous notion that 5S rRNA is a resilient molecule. An analysis of naturally occurring 5S rRNA variants suggests that this molecule may participate in posttranscriptional regulation of gene expression via the nonsense-mediated mRNA decay (NMD) pathway. Lastly, a random mutagenesis analysis of ribosomal protein L10 has resulted in the creation of a powerful toolbox that will be used for elucidation of ribosome export/maturation pathways. Future structure/functional analyses of these mutants may also help to reveal roles of helices 38 and 89 of 25S rRNA.