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School of Biological Sciences

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https://hdl.handle.net/1853/70750

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Organizational Unit

College of Sciences

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Organizational Unit

Center for the Study of Systems Biology

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https://finding-aids.library.gatech.edu/agents/corporate_entities/1131

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Publication Search Results

Now showing 1 - 10 of 13

RNA ligation by hammerhead ribozymes and DNAzyme in plausible prebiotic conditions

(Georgia Institute of Technology, 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.
Binding properties of Hfq to RNA and genomic DNA and the functional implications

(Georgia Institute of Technology, 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.
Characterization of E coli Hfq structure and its RNA binding properties

(Georgia Institute of Technology, 2005-12-07) Sun, Xueguang

Hfq is a bacterial RNA-binding protein recently shown to contain the Sm motif, a characteristic of Sm proteins that function in RNA processing in archaea and eukaryotes. Hfq plays a major role in RNA-RNA interactions regulating translation. Comparative structural modeling and amino acid sequence alignment were used to predict the 3-D structure of Hfq and the model was in excellent agreement with the crystal structure which determined for S. aureus Hfq. The evolution of Hfq was explored by a BLAST search of microbial genomes followed by phyletic analysis. About half of the genomes examined contain at least one gene coding for Hfq. The presence and absence of Hfq closely followed major bacterial clades. The potential RNA binding residues on the two surfaces of the Hfq hexamer were proposed based on the bioinformatics studies and the mutant Hfq proteins with either single or double mutations on the two surfaces of the Hfq hexamer were generated. Their RNA binding properties was biophysically studied by gel-shift assay, fluorescence anisotropy and fluorescence quenching techniques. Results indicated that 1) point mutations on the distal surface of the Hfq hexamer, Y25A and K31A, have a major effect on A18 binding. Both reduce binding by about 1000 fold. Mutations on the proximal surface have a small or no influence on A18 binding. 2) Two mutations, F39A and R16A, on the proximal surface of the Hfq structure reduce binding to the DsrA domain II by 10 fold. Other mutations reduce binding by less than 2 fold. 3) An amino acid covariance was observed in L12 and F39. Mutation L12F can partially restore F39A in DsrA RNA binding. 4) It appears that two Hfq hexamers cooperatively bind one RNA for both DsrADII and A18.
Thermodynamic studies of tandem mismatches and other structural elements in Hairpin and duplex nucleic acids

(Georgia Institute of Technology, 2003-12-01) Bourdelat-Parks, Brooke Nicole
Targeted degradation of RNA by RNase H using stable DNA hairpin oligomers and a study on the effect of temperature and divalent cations on RNA conformational states

(Georgia Institute of Technology, 2002-12) Li, Jing
The stabilities of RNA and DNA structural elements

(Georgia Institute of Technology, 1998-05) Zhu, Jian
DNA complexes with adjacent duplex and triplex domains : thermal denaturation and gel mobility shift analysis

(Georgia Institute of Technology, 1997-12) Nam, Kang Hoon
Characterization of developmentally regulated and lens nuclear proteins binding to upstream sequences of the MP19 LIM2 gene

(Georgia Institute of Technology, 1996-05) Xu, Heng
Determination of non-watson-crick base pair stability and development of a new method for mutation detection

(Georgia Institute of Technology, 1994-12) Ke, Song-Hua
Fermentation of D-xylose and mixtures of D-xylose and glucose by Candida shehatae

(Georgia Institute of Technology, 1993-12) Kastner, James Robert