Co-Expression and Co-Purification of Archaeal and Eukaryal Box C/D RNPs |
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Authors: | Yu Peng Ge Yu Shaoxiong Tian Hong Li |
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Affiliation: | 1. Institute of Molecular Biophysics, Florida State University, Tallahassee, Florida, United States of America.; 2. Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida, United States of America.; Max-Planck-Institute for Terrestrial Microbiology, Germany, |
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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. |
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