Defense Date

4-11-2006

Graduation Date

Spring 2006

Availability

Immediate Access

Submission Type

thesis

Degree Name

MS

Department

Chemistry and Biochemistry

Committee Chair

Rita Mihailescu

Committee Member

Charles T. Dameron

Committee Member

Jeffry D. Madura

Keywords

circular dichroism spectroscopy, RNA melting curves

Abstract

Fragile X syndrome, the most common form of inherited mental retardation, is the result of an unstable expansion of the CGG trinucleotide repeat in the 5' untranslated region of the fragile X mental retardation-1 (FMR1) gene. The abnormal hypermethylation of these CGG repeats causes the transcriptional silencing of the FMR1 gene, and consequently the loss of the fragile X mental retardation protein (FMRP). FMRP is an RNA binding protein that has been shown to use its RGG box domain to bind to G quartet forming RNA. The RNA encoding for the microtubule associated protein 1B (MAP1B), a protein that is involved in neuritic extension and neuronal development, has been identified as a potential FMRP mRNA target. In this study, a thermodynamic and structural analysis of MAP1B RNA and its interactions with the FMRP RGG box was performed. It was determined that MAP1B RNA forms an intramolecular parallel G quartet structure. In the presence of 150 mM KCl, MAP1B RNA appears to fold into two monomeric conformations; however, only one is bound by the FMRP RGG box. A careful study was performed to identify the conditions in which MAP1B RNA folds as a single species and it was determined by NMR spectroscopy and native gel electrophoresis that at low salt concentrations (10 mM KCl) only the MAP1B RNA conformation that is bound by the FMRP RGG box is dominant.

This study also investigated the interactions between MAP1B RNA and the FMRP RGG box. Using fluorescence spectroscopy, it was determined that the FMRP RGG box binds with high affinity to MAP1B RNA. Circular dichroism and UV spectroscopy were employed to determine if the binding of the FMRP RGG peptide changes the structure and the stability of the MAP1B RNA G quartet structure.

Format

PDF

Language

English

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