Crystallization of RNA-protein complexes: from synthesis and purification of individual components to crystals.
A broad range of biological processes relies on complexes between RNA and proteins. Crystallization of RNA-protein complexes can yield invaluable information on structural organizations of key elements of cellular machinery. However, crystallization of RNA-protein complexes is often challenging and requires special approaches. Here we review the purification of RNA, RNA-binding proteins, and the formation and crystallization of RNA-protein complexes, using the crystallization of the P3 RNA domain of ribonuclease MRP, a multicomponent ribonucleoprotein complex involved in the metabolism of various RNA molecules, as an example. The RNA-protein complex was formed using gel-purified RNA, produced by run-off transcription with T7 RNA polymerase in vitro, and proteins that were overexpressed in Escherichia coli and purified to be RNase-free. The complex was crystallized using a sitting drop setup; initial screening for suitable crystallization conditions was performed using a sparse matrix approach.
Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA, USA.
Anna
A
Perederina
Andrey S
AS
Krasilnikov
D005591:Chemical Fractionation / Q000379:methods*; D003460:Crystallization / Q000379:methods*; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D009841:Oligonucleotides / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D012313:RNA / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D012260:Ribonucleases / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D014158:Transcription, Genetic; D015003:Yeasts / Q000201:enzymology
D009841:Oligonucleotides; C039919:RNA, recombinant; D012313:RNA; D004247:DNA; D012260:Ribonucleases