Poster Presentation The 43rd Lorne Conference on Protein Structure and Function 2018

Characterization and redesign of pentatricopeptide repeat (PPR) - RNA binding proteins (#162)

Kalia Bernath-Levin 1 , Bernard Gutmann , Ian Small
  1. University of Western Australia, Crawley, WA, Australia

Pentatricopeptide repeat (PPR) proteins are sequence-specific RNA binding proteins located in organelles that are involved in broad range of post-transcriptional RNA maturation processes. The plant PPR proteins are grouped into P and PLS classes which generally fulfil distinct functions. The majority of the PLS-class PPR proteins function as editing factors. P-class PPR proteins act in defining 5' and 3' RNA termini, splicing, and promoting or blocking initiation of translation. Given their role in determining the fate of RNA transcripts, they are good candidates for proteins that can be redesigned for binding RNA of interest. Unlike other RNA- or DNA-binding proteins that are currently tested as a scaffold for protein redesign, PPRs are highly specific. The recently developed “PPR code” suggests that it might be possible to redesign the PPR protein to recognize a particular RNA target based on the identity of two key amino acids. Crystal structures, analysis of native PPR sequences as well as binding assays indicate that more amino acids influence binding affinity and specificity. To understand the potential role of various amino acids in binding affinity and specificity, we have devised a 96-well screening method that offers to test binding of protein-variants to different RNA sequences. The screen is versatile - it can be done in different buffer compositions, with diverse cell-lysates, and also binding to more than one RNA sequences can be tested in parallel in a single well. We will use this method to screen and characterize protein-libraries that focus on selected amino acids.