Partial reconstitution of human RNase P in HeLa cells between its RNA subunit with an affinity tag and the intact protein components.
An RNA affinity tag was incorporated into the RNA subunit of human nuclear RNase P. The tagged RNA assembled with the protein components of RNase P inside HeLa cells to generate an active enzyme. Because of the specificity of the RNA tag to streptavidin, the reconstituted complex could be separated from the native enzyme and other ribonucleoproteins (particularly RNase MRP) by streptavidin agarose chromatography and could be recovered by the eluting agent, biotin. A mutant, tagged RNase P RNA, whose P3 domain was partially replaced, could not reconstitute with the proteins to yield an active enzyme. The P3 domain, therefore, is critical for the structure and function of RNase P.
Department of Molecular, Cellular and Developmental Biology, Yale University, 266 Whitney Avenue, New Haven, CT 06511, USA.
Yong
Y
Li
Sidney
S
Altman
D000345:Affinity Labels / Q000737:chemistry*; D015152:Blotting, Northern; D015153:Blotting, Western; D002847:Chromatography, Agarose / Q000379:methods; D003001:Cloning, Molecular; D004722:Endoribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D009154:Mutation; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000235:genetics; D012313:RNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D043262:Ribonuclease P; D012261:Ribonucleoproteins / Q000378:metabolism*; D019809:Streptavidin / Q000737:chemistry; D014162:Transfection
D000345:Affinity Labels; D016337:RNA, Catalytic; D012261:Ribonucleoproteins; D012313:RNA; D019809:Streptavidin; D004722:Endoribonucleases; C468368:RPP14 protein, human; D043262:Ribonuclease P