Purification of RNA and RNA-protein complexes by an R17 coat protein affinity method.
We describe an affinity chromatography method to isolate specific RNAs and RNA-protein complexes formed in vivo or in vitro. It exploits the highly selective binding of the coat protein of bacteriophage R17 to a short hairpin in its genomic RNA. RNA containing that hairpin binds to coat protein that has been covalently bound to a solid support. Bound RNA-protein complexes can be eluted with excess R17 recognition sites. Using purified RNA, we demonstrate that binding to immobilized coat protein is highly specific and enables one to separate an RNA of interest from a large excess of other RNAs in a single step. Surprisingly, binding of an RNA containing non-R17 sequences to the support requires two recognition sites in tandem; a single site is insufficient. We determine optimal conditions for purification of specific RNAs by comparing specific binding (retention of RNAs with recognition sites) to non-specific binding (retention of RNAs without recognition sites) over a range of experimental conditions. These results suggest that binding of immobilized coat protein to RNAs containing two sites is cooperative. We illustrate the potential utility of the approach in purifying RNA-protein complexes by demonstrating that a U1 snRNP formed in vivo on an RNA containing tandem recognition sites is selectively retained by the coat protein support.
Bardwell
V J
VJ
Cell and Molecular Biology Program, University of Wisconsin-Madison 53706.
Wickens
M
M
D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D002213:Capsid / Q000235:genetics*; D036022:Capsid Proteins; D002846:Chromatography, Affinity; D003090:Coliphages / Q000235:genetics*; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009865:Oocytes / Q000737:chemistry; D012313:RNA / Q000737:chemistry*; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins / Q000235:genetics* / Q000302:isolation & purification; D014981:Xenopus
D036022:Capsid Proteins; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; D012313:RNA