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search.filters.applied.f.advisor: Wartell, Roger M. × End date: 2015 × Start date: 2010 × search.filters.applied.f.isOrgUnitOfPublicationTitle: College of Sciences × search.filters.applied.f.isOrgUnitOfPublicationTitle: School of Biological Sciences × search.filters.applied.f.resourcesubtype: Dissertation ×

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RNA ligation by hammerhead ribozymes and DNAzyme in plausible prebiotic conditions

2015-08-21 , Lie, Lively

This work is focused on the ligation activity of the hammerhead ribozyme and DNAzymes in plausible prebiotic conditions. Before the Great Oxidation Event, RNA may have interacted with soluble Fe2+, as a replacement or in combination with Mg2+. Divalent metal cations are sometimes necessary in ribozyme activity by interacting with mostly phosphates to influence the tertiary structure of an RNA. In some cases, these metal cations help in the acid/base chemistry in catalytic cores. Chapter 2 reveals the benefits and drawbacks of hammerhead ribozyme ligation with Fe2+. Both ligation and cleavage of the hammerhead is enhanced, but an unexpected problem arose, RNA aggregation that is difficult to denature. Chapter 3 and 4 focuses on the hammerhead ligation in ice. Freeze-induced ligation frees the hammerhead from divalent metal requirements and when combined with heat-freeze cycles to mimic day and night, yield reaches 60%. Freezing the reaction mixture also reduces sequence specificity between enzyme and substrates. Chapter 5 reveals a RNA-cleaving DNAzyme that can ligate cleaved RNA substrates when the reaction mixture is frozen. The significance behind this chapter is that previous ligating DNAzymes require high-energy triphosphates and instead uses a 2’3’-cyclic phosphate. This 2’3’-cyclic phosphate is already a product of the cleavage reaction of the DNAzyme and the cleavage/ligation reaction is in effect recycling the same materials.

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Binding properties of Hfq to RNA and genomic DNA and the functional implications

2011-05-10 , Updegrove, Taylor Blanton

The bacterial RNA binding protein Hfq is a key component for bacterial sRNA mediated riboregulation of mRNA expression. A kinetic and thermodynamic analysis of Hfq binding to its sRNA targets DsrA, RprA, and OxyS, and to its mRNA target rpoS was carried out. The ability of Hfq to significantly enhance the stability of the DsrA-rpoS and RprA-rpoS complex was demonstrated, and the entire untranslated leader region of rpoS was shown to be important for Hfq binding and in Hfq facilitated sRNA-mRNA duplex formation. Hfq was not shown to enhance OxyS binding to rpoS. DsrA and OxyS were shown to bind mostly to the proximal surface region of Hfq, while RprA bound to both proximal and distal surface regions. The rpoS leader region was shown to possess at least two distinct Hfq binding sites, with one site binding the proximal region and the other to the distal region of Hfq. These sites were shown to be important for Hfq to stimulate DsrA-rpoS binding. The outer-circumference region and the C-terminal tail of Hfq does not play a major role in binding DsrA, RprA, OxyS and rpoS, and in stimulating DsrA-rpoS binding. Evidence was obtained implicating Hfq to bind DsrA, RprA, OxyS, and oligo rA18 in a 1:1 protein to RNA stoichiometry. Binding properties of Hfq to E. coli genomic DNA were examined. Double stranded DNA was shown to bind mostly on the distal surface region and the C-terminal tail of Hfq with an affinity 10 fold less than Hfq targeted RNA. Single stranded DNA binds Hfq more tightly than double stranded DNA and binding seems to be sequence specific. Evidence indicates Hfq binds certain sequences of the E. coli genome.

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