Investigations of the Mechanisms and Applications of Pentatricopeptide Repeat (PPR) Proteins

Abstract

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This dissertation contains unpublished co-authored material.