| Abstrakt: |
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 inEscherichia 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. |
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