In vitro reconstitution and affinity purification of catalytically active archaeal box C/D sRNP complexes.
Archaeal box C/D RNAs guide the site-specific 2'-O-methylation of target nucleotides in ribosomal RNAs and tRNAs. In vitro reconstitution of catalytically active box C/D RNPs by use of in vitro transcribed box C/D RNAs and recombinant core proteins provides model complexes for the study of box C/D RNP assembly, structure, and function. Described here are protocols for assembly of the archaeal box C/D RNP and assessment of its nucleotide modification activity. Also presented is a novel affinity purification scheme that uses differentially tagged core proteins and a sequential three-step affinity selection protocol that yields fully assembled and catalytically active box C/D RNPs. This affinity selection protocol can provide highly purified complex in sufficient quantities not only for biochemical analyses but also for biophysical approaches such as cryoelectron microscopy and X-ray crystallography.
Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC, USA.
Keith
K
Gagnon
Xinxin
X
Zhang
E Stuart
ES
Maxwell
D002846:Chromatography, Affinity / Q000379:methods*; D002852:Chromatography, Ion Exchange / Q000379:methods; D017016:Methanococcaceae; D017411:Ribonucleoproteins, Small Nuclear / Q000302:isolation & purification*
D017411:Ribonucleoproteins, Small Nuclear