Purification of functional RNA-protein complexes using MS2-MBP.
Biological machines composed of RNAs and proteins play essential roles in many biological processes. To better understand the mechanism and function of these machines, it is critical to isolate them in a highly purified and functional form. A method for isolating functional RNA-protein complexes assembled in vitro is described. The approach combines gel filtration and an affinity-chromatography strategy using the bacteriophage MS2 coat protein, which binds to a specific RNA-hairpin structure. Using this method, highly purified and functional human spliceosomes have been isolated. The purified spliceosome preparation is used to determine the protein components of the spliceosome by mass spectrometry and to examine the structure of the spliceosome by electron microscopy.
Zhou
Zhaolan
Z
Children's Hospital and Harvard Medical School, Boston, Massachusetts, USA.
Reed
Robin
R
D001665:Binding Sites; D036022:Capsid Proteins / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002474:Cell-Free System; D002846:Chromatography, Affinity / Q000379:methods*; D002850:Chromatography, Gel / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D007202:Indicators and Reagents; D017909:Levivirus; D058619:Maltose-Binding Proteins; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D012322:RNA Precursors / Q000302:isolation & purification / Q000378:metabolism; D012342:RNA, Small Nuclear / Q000302:isolation & purification / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012261:Ribonucleoproteins / Q000302:isolation & purification* / Q000378:metabolism; D017381:Spliceosomes / Q000737:chemistry*; D013379:Substrate Specificity
D036022:Capsid Proteins; D002352:Carrier Proteins; D007202:Indicators and Reagents; D058619:Maltose-Binding Proteins; D012322:RNA Precursors; D012342:RNA, Small Nuclear; D011993:Recombinant Fusion Proteins; D012261:Ribonucleoproteins; D012313:RNA