Abstract
Transcription factor hTFIIIC was purified from cytoplasmic extracts of HeLa cells using four different chromatographic steps. This procedure yields a protein fraction which actively supports transcription in reconstitution assays and contains five major polypeptide chains with a molecular mass ranging from 25 to 250 kDa as estimated by SDS-PAGE and silver staining. In this fraction a polypeptide with a molecular mass of approximately 110 kDa could be identified as a specific DNA-binding component of hTFIIIC. By electrophoretic mobility shift and footprinting analyses it could be demonstrated that purified hTFIIIC binds specifically to the 5S gene. The protected region encompasses the A-Box promoter element and flanking sequences extending toward the 5'-proximal end of the gene. By addition of hTFIIIC to preformed TFIIIA/5S DNA complexes, we observe an additive effect of both factors on the footprint boundaries.
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Selected References
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