A FACS-based screening strategy to assess sequence-specific RNA-binding of Pumilio protein variants in E. coli.
Sequence-specific and programmable binding of proteins to RNA bears the potential to detect and manipulate target RNAs. Applications include analysis of subcellular RNA localization or post-transcriptional regulation but require sequence-specificity to be readily adjustable to any target RNA. The Pumilio homology domain binds an eight nucleotide target sequence in a predictable manner allowing for rational design of variants with new specificities. We describe a high-throughput system for screening Pumilio variants based on fluorescence-activated cell sorting of E. coli. Our approach should help optimizing variants obtained from rational design regarding folding and stability or identifying new variants with alternative binding modes.
Stefanie J
SJ
Kellermann
Andrea
A
Rentmeister
D001483:Base Sequence; D004926:Escherichia coli / Q000166:cytology* / Q000378:metabolism; D005434:Flow Cytometry; D009154:Mutation; D011485:Protein Binding; D012313:RNA / Q000235:genetics* / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D013379:Substrate Specificity
D016601:RNA-Binding Proteins; D012313:RNA