Barkan, AliceMiranda, Rafael2018-04-102018-04-10https://hdl.handle.net/1794/23135Pentatricopeptide 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 This dissertation contains previously published and unpublished co-authored material.en-USAll Rights Reserved.Electronic Thesis or Dissertation