Access Type

Open Access Dissertation

Date of Award

1-1-2011

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Chemistry

First Advisor

David Rueda

Abstract

In the cell, RNA and protein, interact to form ribonucleoprotein complexes (RNPs) that have vital structural, catalytic and regulatory roles. Despite their functional importance, the mechanistic details and dynamics of RNPs are poorly understood. Single-molecule Fluorescence Resonance Energy Transfer (smFRET) techniques that provide information about heterogeneity and dynamic behaviors of molecules have been developed to investigate inter- and intra-molecular interactions. Here we have used FRET in combination with smFRET to study three very different RNP systems.

Alternative splicing is a highly regulated biological process that plays a crucial role in proteomic diversity in eukaryotes. One splicing regulator, PTB, has been proposed to repress splicing by looping RNA between two binding sites. Here, we examined the looping activity of a minimal PTB construct (PTB34) on various RNA oligonucleotides and found that PTB34 requires at least a 15 nucleotide linker between binding sites for efficient looping. A PTB antagonist, Fox-1, has been hypothesized to compete with PTB to reduce looping and promote exon inclusion. Our data suggest that Fox-1 indeed disrupts PTB binding and looping, supporting the hypothesis that Fox-1 breaks RNA looping to enhance splicing of alternative exons.

Interactions between ribosomal proteins and ribosomal RNA facilitate the formation of functional ribosomes. Studies of a central junction in 16S rRNA and the primary binding protein that triggers a conformational change, S15, show that mutations that alter the junction dynamics affect 30S assembly. Although partially functional mutants are complemented by over-expression of S15, nonfunctional mutants are not. Comparison of the structural dynamics of these mutants and WT sequence in the presence and absence of S15 revealed specific sequence and structural motifs in the junction that are important for ribosome function.

Small non-coding RNAs regulate gene expression in response to biological stimuli through a mechanism that relies on changes in RNA-RNA interactions, for instance switching between two hairpins and an extended duplex. In the cell, proteins, such as Hfq, facilitate the formation of functional RNA structures. Here we show that Hfq acts as a chaperone to overcome high-energy barriers and promote the progression of kissing hairpins through strand displacement to an extended duplex formation.

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