Date of Award

2010

Degree Type

Dissertation

Degree Name

PhD. Chemistry

Department

Chemistry and Biochemistry

Advisor

Stephen P. Kelty

Committee Member

Cecilia H. Marzabadi

Committee Member

Wyatt R. Murphy

Committee Member

Cosimo Antonacci

Committee Member

George J. Turner

Keywords

Pure sciences, Nucleic acids, Secondary structures, Gene expression

Abstract

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.

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