Theses and Dissertations from UMD
Permanent URI for this communityhttp://hdl.handle.net/1903/2
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
More information is available at Theses and Dissertations at University of Maryland Libraries.
Browse
5 results
Search Results
Item Ribosomal Protein L11: A Cog in the Nanomachine(2011) Rhodin, Michael Hoover Johannes; Dinman, Jonathan D; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Comprised of two major subunits of both rRNA and proteins, the ribosome is a biological nanomachine, acting as the central player in the process of protein translation. Recent advances in molecular imaging have enabled the visualization of the disparate functional centers within the ribosome, leading to the question of how these critical regions coordinate their actions and communicate with each other. This work examines the essential ribosomal protein L11, located in the central protuberance of the large subunit. L11 maintains connections with the 5S rRNA, H84 of the 25S rRNA, comes in close proximity to the T-loop of the bound peptidyl tRNA, and shares an intersubunit bridge with small subunit protein S18. L11 was found to have a critical dynamic loop which samples the occupancy status of the P-site pocket of the ribosome and communicates this information through H84. L11's intersubunit bridge (the B1b/c bridge) mediates an intersubunit communication network from the decoding center to the peptidyl transferase center of the ribosome. L11 is also involved in proper subunit joining. Mutations in L11 were found to have effects on A- and P-site tRNA binding, translational fidelity, and growth and viability of yeast cells.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 Ribosome integrity and translational fidelity require accurate modification and processing of rRNA in the yeast Saccharomyces cerevisiae(2006-11-21) Roshek, Jennifer Lynn Baxter; Dinman, Jonathan D; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Translating mRNA sequences into functional proteins is a fundamental process necessary for the viability of organisms throughout all kingdoms of life. The ribosome carries out this process with a delicate balance between speed and accuracy. Although kinetic and biochemical studies along with high resolution crystal structures have provided much information about the ribosome, many of the underlying mechanisms of ribosome function are still poorly understood. This work seeks to understand how ribosome structure and function are affected by changes in rRNA as caused by two very different mechanisms. mof6-1, originally isolated as a recessive mutation which promoted increased efficiencies of programmed -1 ribosomal frameshifting, was found to be an allele of RPD3 which encodes a histone deacetylase that is involved in transcriptional activation and silencing. This mutant demonstrated a delay in ribosomal RNA (rRNA) processing leading to changes in reading frame maintenance and ribosomal A-site specific defects. To understand the role of cis-acting changes to rRNA, yeast strains deficient in rRNA modifications in the peptidyl transferase center of the ribosome were monitored for changes in ribosome structure and translational fidelity. Analyses revealed mutant phenotypes including sensitivity to translational inhibitors; changes in reading frame maintenance, nonsense suppression and aa-tRNA selection; and increased rates of A-site tRNA binding to the mutant ribosome. One mutant in particular, spb1DA/snr52Δ, promoted increased rates of programmed -1 ribosomal frameshifting, increased rates of near cognate tRNA selection and A-site tRNA binding. Structural analysis of spb1DA/snr52Δ revealed changes consistent with a more accessible ribosomal A-site. These results suggest that rRNA nucleotide modifications produce small but distinct changes in ribosome structure and function contributing to overall translational fidelity. Taken together, these data suggest that rRNA, a main component of the ribosome, contributes directly to translational fidelity. Defects in rRNA caused by changes in both its processing and modification can cause changes in reading frame maintenance, nonsense suppression, aa-tRNA selection and binding as well as ribosome structure.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.Item PRELIMINARY GENETIC CHARACTERIZATION OF RIBOSOMAL PROTEIN L10 IN SACCHAROMYCES CEREVISIAE(2005-05-27) Simmons, Mary Kecia Rigsby; Dinman, Jonathan D; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)While recent research has focused on the exciting concept of the ribosome as a ribozyme, ribosomal proteins have been largely overlooked. This study focuses on four previously identified mutants of the ribosomal large subunit protein L10, in an effort to better understand how the protein's structure corresponds to its function. All of the mutants studied displayed how the embedded nature of L10 caused only slight conformational changes to impact function of the ribosome as a whole; in these cases primarily altering the interaction between the ribosomal A-site and aminoacyl-tRNAs.