Chemistry Theses and Dissertations

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https://hdl.handle.net/1794/2046

This collection contains some of the theses and dissertations produced by students in the University of Oregon Chemistry Graduate Program. Paper copies of these and other dissertations and theses are available through the UO Libraries.

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Browsing Chemistry Theses and Dissertations by Author "Barkan, Alice"

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    Investigations of the Mechanisms and Applications of Pentatricopeptide Repeat (PPR) Proteins
    (University of Oregon, 2019-04-30) McDermott, James; Barkan, Alice
    Pentatricopeptide repeat proteins (PPR) proteins are helical-repeat proteins that bind RNA in a modular one-nucleotide:one-repeat fashion. The specificity of a given PPR repeat is dictated by amino acids at two-positions, which recognize a particular nucleotide through hydrogen bonds with the Watson-Crick face. The combinations of amino acids at these positions that give rise to nucleotide specificity is referred to as the PPR-code. The modular and programmable nature of PPR proteins makes them promising candidates for use in applications that require targeting a protein to a specific RNA sequence. One mechanism by which PPR proteins act involves the remodeling of inhibitory RNA hairpins that sequester a ribosome binding site upstream of the gene. However, other evidence suggests that PPR protein-RNA interactions can be inhibited by RNA secondary structure. It is not clear what parameters determine which partner prevails in binding to the RNA. I investigated how the position and strength of an RNA structure impacts PPR:RNA binding and determined that even weak RNA structures are able to inhibit PPR:RNA binding. Additionally, I investigated the driving forces of PPR:RNA binding kinetics. Together, these parameters will benefit the design of synthetic PPR proteins for specific purposes. Several groups have demonstrated that synthetic PPR proteins can be designed to bind a specified RNA sequence in vitro. However, no work has been performed using engineered, or designer PPR proteins in an in vivo setting. I demonstrated the feasibility of using a designer PPR protein to bind a specified RNA in vivo, and I applied this capability for a specific application – the purification of an endogenous ribonucleoprotein particle to identify associated proteins. This dissertation contains unpublished co-authored material.
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    Sequence Specific RNA Recognition by Pentatricopeptide Repeat Proteins: Beyond the PPR Code
    (University of Oregon, 2018-04-10) Miranda, Rafael; Barkan, Alice
    Pentatricopeptide repeat (PPR) proteins are helical-repeat proteins that bind RNAs through a simple 1-repeat:1-nucleotide manner. Nucleotide specificity is determined by an amino acid code, the PPR code. This modular interaction mode, predictable code for nucleotide specificity, and simple repeating architecture make them a promising scaffold for engineering proteins to bind custom RNA sequences and binding site prediction of native PPR proteins. Despite these features, the alignments of the binding sites of well-characterized PPR proteins to the predicted binding sites often have mismatches and discontinuities, suggesting a tolerance for mismatches. In order to maximize the ability to predict the binding sites of native PPR proteins and effectively generate designer PPR proteins with predictable specificity, it will be important to address how affinity and specificity is distributed across a PPR tract. I developed a high- throughput bind-n-seq technique to rapidly and thoroughly address these questions. The affinity and specificity of the native PPR protein, PPR10 was determined using bind-n- seq. The results demonstrate that not all of PPR10’s repeats contribute equally to binding affinity, and there were sequence specific interactions that could not be explained by the PPR code, suggesting alternate modes of nucleotide recognition. A similar analysis of four different designer PPR proteins showed that they recognize RNA according to the code and lacked any alternate modes of nucleotide recognition, implying that the non- canonical sequence specific interactions represent idiosyncratic features of PPR10. This analysis also showed that N-terminal and purine specifying repeats have greater contributions to binding affinity, and that longer scaffolds have a greater tolerance for mismatches. Together, these findings highlight the challenges for binding site prediction and present implications for the design of PPR proteins with minimum off-target binding. This dissertation contains previously published and unpublished co-authored material.