STRUCTURAL ANALYSIS OF RIBOSOME BINDING ELEMENTS OF THE 3´ UTR IN TWO POSITIVE-STRAND PLANT RNA VIRUSES

Turnip crinkle virus (TCV) and Pea enation mosaic virus (PEMV) are two positive (+)-strand RNA viruses that are used to investigate the regulation of translation and replication due to their small size and simple genomes. Both viruses contain cap-independent translation elements (CITEs) within their 3´ untranslated regions (UTRs) that fold into tRNA-shaped structures (TSS) according to nuclear magnetic resonance and small angle x-ray scattering analysis (TCV) and computational prediction (PEMV). Specifically, the TCV TSS can directly associate with ribosomes and participates in RNA-dependent RNA polymerase (RdRp) binding. The PEMV kissing-loop TSS (kl-TSS) can simultaneously bind to ribosomes and associate with the 5´ UTR of the viral genome. Mutational analysis and chemical structure probing methods provide great insight into the function and secondary structure of the two 3´ CITEs. However, lack of 3-D structural information has limited our understanding of their functional dynamics. Here, I report the folding dynamics for the TCV TSS using optical tweezers (OT), a single molecule technique. My study of the unfolding/folding pathways for the TCV TSS has provided an unexpected unfolding pathway, confirmed the presence of Ψ3 and hairpin elements, and suggested an interconnection between the hairpins and pseudoknots. In addition, this study has demonstrated the importance of the adjacent upstream adenylate-rich sequence for the formation of H4a/Ψ3 along with the contribution of magnesium to the stability of the TCV TSS. In my second project, I report on the structural analysis of the PEMV kl-TSS using NMR and SAXS. This study has re-confirmed the base-pair pattern for the PEMV kl-TSS and the proposed interaction of the PEMV kl-TSS with its interacting partner, hairpin 5H2. The molecular envelope of the kl-TSS built from SAXS analysis suggests the kl-TSS has two functional conformations, one of which has a different shape from the previously predicted tRNA-shaped form. Along with applying biophysical methods to study the structural folding dynamics of RNAs, I have also developed a technique that improves the production of large quantities of recombinant RNAs in vivo for NMR study. In this project, I report using the wild-type and mutant E.coli strains to produce cost-effective, site-specific labeled, recombinant RNAs. This technique was validated with four representative RNAs of different sizes and complexity to produce milligram amounts of RNAs. The benefit of using site-specific labeled RNAs made from E.coli was demonstrated with several NMR techniques.

ANALYSIS AND SIMULATION OF ENERGY USE AND COST AT A MUNICIPAL WASTEWATER TREATMENT PLANT

The cost of electricity, a major operating cost of municipal wastewater treatment plants, is related to influent flow rate, power price, and power load. With knowledge of inflow and price patterns, plant operators can manage processes to reduce electricity costs. Records of influent flow, power price, and load are evaluated for Blue Plains Advanced Wastewater Treatment Plant. Diurnal and seasonal trends are analyzed. Power usage is broken down among treatment processes. A simulation model of influent pumping, a large power user, is developed. It predicts pump discharge and power usage based on wet-well level. Individual pump characteristics are tested in the plant. The model accurately simulates plant inflow and power use for two pumping stations [R2 = 0.68, 0.93 (inflow), R2 =0.94, 0.91(power)]. Wet-well stage-storage relationship is estimated from data. Time-varying wet-well level is added to the model. A synthetic example demonstrates application in managing pumps to reduce electricity cost.

Interactive Effects of Plant Species and Organic Carbon on Nitrate Removal in Chesapeake Bay Treatment Wetlands

Nitrate from agricultural runoff are a significant cause of algal blooms in estuarine ecosystems such as the Chesapeake Bay. These blooms block sunlight vital to submerged aquatic vegetation, leading to hypoxic areas. Natural and constructed wetlands have been shown to reduce the amount of nitrate flowing into adjacent bodies of water. We tested three wetland plant species native to Maryland, Typha latifolia (cattail), Panicum virgatum (switchgrass), and Schoenoplectus validus (soft-stem bulrush), in wetland microcosms to determine the effect of species combination and organic amendment on nitrate removal. In the first phase of our study, we found that microcosms containing sawdust exhibited significantly greater nitrate removal than microcosms amended with glucose or hay at a low nitrate loading rate. In the second phase of our study, we confirmed that combining these plants removed nitrate, although no one combination was significantly better. Furthermore, the above-ground biomass of microcosms containing switchgrass had a significantly greater percentage of carbon than microcosms without switchgrass, which can be studied for potential biofuel use. Based on our data, future environmental groups can make a more informed decision when choosing biofuel-capable plant species for artificial wetlands native to the Chesapeake Bay Watershed.