Stability of Nucleic Acid Secondary Structures and Their Contribution to Gene Expression

<p>Bacteriorhodopsin (bR) is a highly expressed transmembrane protein that acts as a light-driven proton pump converting light energy into a proton gradient. The extraordinary levels of expression achieved (15-30 mg per liter of culture) are a result of very efficient biogenesis that originates from molecular information encoded in the bacterio-opsin gene {bop) (1). DNA sequence analysis and predictive folding algorithms suggest that the first twenty-five bases of the bop gene mRNA can form a secondary structural element (a "stem-loop"). Using biophysical methods, the goal was to determine if the stem-loop structure exists in solution conditions that mimic the in vivo biological environment (42°C and 4M KCl) (2-4). The salt-dependent thermal stability of a DNA model of the stem-loop sequence was obtained using temperature-controlled UVabsorption and Circular Dichroism (CD) spectroscopy and Differential Scanning Calorimetry (DSC). The combinatorial analysis indicates that an energetically favorable stem-loop structure forms in all solution conditions examined. The stem-loop structure is stabilized at very high salt concentrations, a requirement for a role in bop gene expressivity (AG= -1.13 kcal/mol, AH= -16.78 kcal/mol, AS= - 49.67 cal/mol/K). While the Tm values evaluated by all three physical techniques were comparable, comparison of the calorimetrie and van't Hoff enthalpies indicate that the folding mechanism is not a two-state process. SVD analysis of the UV and CD spectral transitions confirm that formation of the stem-loop structure proceeds through a single significant folding intermediate. Similar analysis of the RNA sequence demonstrates that an RNA stem- loop structure can form and is significantly more stable than that of the DNA structure at all salt concentrations evaluated.</p>

Name thesis:degree_name

PhD. Chemistry

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Chemistry and Biochemistry

Year dc:date.available

2010

Author dc:creator

• Darwish, Maged A

Contributors dc:contributor

• Stephen P. Kelty
• Cecilia H. Marzabadi
• Wyatt R. Murphy
• Cosimo Antonacci
• George J. Turner

• Pure sciences
• Nucleic acids
• Secondary structures
• Gene expression
• Biochemistry

Repository record dc:identifier

https://scholarship.shu.edu/dissertations/1435

OAI identifier oai:identifier

oai:scholarship.shu.edu:dissertations-2449