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. 1989 Jul 11;17(13):5003–5016. doi: 10.1093/nar/17.13.5003

Purification of human transcription factor IIIC and its binding to the gene for ribosomal 5S RNA.

H R Schneider 1, R Waldschmidt 1, D Jahn 1, K H Seifart 1
PMCID: PMC318090  PMID: 2762117

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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