title: Co-expression and co-purification of archaeal and eukaryal box C/D RNPs.
abstract: Box C/D ribonucleoprotein particles (RNPs) are 2'-O-methylation enzymes required for maturation of ribosomal and small nuclear RNA. Previous biochemical and structural studies of the box C/D RNPs were limited by the unavailability of purified intact RNPs. We developed a bacterial co-expression strategy based on the combined use of a multi-gene expression system and a tRNA-scaffold construct that allowed the expression and purification of homogeneous archaeal and human box C/D RNPs. While the co-expressed and co-purified archaeal box C/D RNP was found to be fully active in a 2'-O-methylation assay, the intact human U14 box C/D RNP showed no detectable catalytic activity, consistent with the earlier findings that assembly of eukaryotic box C/D RNPs is nonspontaneous and requires additional protein factors. Our systems provide a means for further biochemical and structural characterization of box C/D RNPs and their assembly factors.
authors: Institute of Molecular Biophysics, Florida State University, Tallahassee, Florida, United States of America.
authors: Yu
authors: Y
authors: Peng
authors: Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida, United States of America.
authors: Ge
authors: G
authors: Yu
authors: Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida, United States of America.
authors: Shaoxiong
authors: S
authors: Tian
authors: Institute of Molecular Biophysics, Florida State University, Tallahassee, Florida, United States of America; Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida, United States of America.
authors: Hong
authors: H
authors: Li
mesh_terms: D001105:Archaea / Q000378:metabolism*; D019843:Archaeal Proteins / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000302:isolation & purification* / Q000378:metabolism*
chemical_list: D019843:Archaeal Proteins; D012261:Ribonucleoproteins