Abstract:
:Pentatricopeptide repeat (PPR) proteins are helical-repeat proteins that offer a promising scaffold for the engineering of proteins to bind specified RNAs. PPR tracts bind RNA in a modular 1-repeat, 1-nucleotide fashion. An amino acid code specifying the bound nucleotide has been elucidated. However, this code does not fully explain the sequence specificity of native PPR proteins. Furthermore, it does not address nuances such as the contribution toward binding affinity of various repeat-nucleotide pairs or the impact of mismatches between a repeat and aligning nucleotide. We used an in vitro bind-n-seq approach to describe the population of sequences bound by four artificial PPR proteins built from consensus scaffolds. The specificity of these proteins can be accounted for by canonical code-based nucleotide recognition. The results show, however, that interactions near the 3'-end of binding sites make less contribution to binding affinity than do those near the 5'-end, that proteins with 11 and 14 repeats exhibit similar affinity for their intended targets but 14-repeats are more permissive for mismatches, and that purine-binding repeats are less tolerant of transversion mismatches than are pyrimidine-binding motifs. These findings have implications for mechanisms that establish PPR-RNA interactions and for optimizing PPR design to minimize off-target interactions.
journal_name
Nucleic Acids Resjournal_title
Nucleic acids researchauthors
Miranda RG,McDermott JJ,Barkan Adoi
10.1093/nar/gkx1288subject
Has Abstractpub_date
2018-03-16 00:00:00pages
2613-2623issue
5eissn
0305-1048issn
1362-4962pii
4780162journal_volume
46pub_type
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