Expression and purification of recombinant hRPABC25, hRPABC17, and hRPABC14.4, three essential subunits of human RNA polymerases I, II, and III.
Transcription of eukaryotic genes is performed by RNA polymerases I, II, and III, which synthesize ribosomal, messenger, and transfer RNAs, respectively. Eukaryotic RNA polymerases are large macromolecular complexes composed of multiple subunits. Among these subunits, five are shared by all RNA polymerases and are essential for cell growth and viability. Remarkably, the human common subunits are structurally conserved and functionally interchangeable with their yeast homologues and are believed to play an important role in the assembly of the three transcription complexes. To understand the structure and function of human RNA polymerases, we overexpressed the common subunits hRPABC25, hRPABC17, and hRPABC14.4 as hexahistidine fusions in Escherichia coli. The recombinant proteins were purified using metal-chelate affinity chromatography on Ni-NTA resin and gel filtration. Depending on the subunit, the yield was 5-17 mg of purified recombinant protein per liter of culture medium. The purified proteins were of high quality and sufficient quantity for structural studies, as demonstrated by the successful crystallization of hRPABC17 and hRPABC14.4. The expression and purification of the common subunits hRPABC25, hRPABC17, and hRPABC14. 4 will make possible their structural analysis with X-ray crystallography and nuclear magnetic resonance, providing important insights into the structure and function of the three human RNA polymerases.
Hiremath
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CN
Division of Experimental Medicine, Harvard Medical School, Boston, Massachusetts, 02115, USA.
Ladias
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JA
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003460:Crystallization; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D006639:Histidine; D006801:Humans; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D012318:RNA Polymerase I / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D012319:RNA Polymerase II / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D012320:RNA Polymerase III / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification
D010455:Peptides; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C033223:polyhistidine; D006639:Histidine; D012319:RNA Polymerase II; D012318:RNA Polymerase I; D012320:RNA Polymerase III