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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.

SUBMITTER: Peng Y

PROVIDER: S-EPMC4117494 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Co-expression and co-purification of archaeal and eukaryal box C/D RNPs.

Peng Yu Y Yu Ge G Tian Shaoxiong S Li Hong H

PloS one 20140731 7

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- ...[more]

PMID: 25078083

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