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

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 hThlllC. By electrophoretic mobility shift and footprinting analyses it could be demonstrated that purified hTFlllC 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 hTFlIIC to preformed TFUIA≤5S DNA complexes, we observe an additive effect of both factors on the footprint boundaries.