title: Purification and characterization of two forms of human transcription factor IIIC.
abstract: We showed previously that HeLa cell nuclear extracts contain two forms of transcription factor IIIC (TFIIIC) that formed chromatographically distinct TFIIIC-promoter complexes (Hoeffler, W. K., Kovelman, R., and Roeder, R. G. (1988) Cell 53, 907-920). One of these forms, the upper-band form, correlated with TFIIIC transcriptional activity, whereas the lower-band form bound to the VA1 promoter but supported little or no transcriptional activity. Using both transcription and DNA-binding assays, we have now purified both the upper-band form and the lower-band form of TFIIIC to near-homogeneity. The upper-band form is composed of five polypeptides with estimated sizes of 220, 110, 102, 90, and 63 kDa. The largest of these polypeptides can be cross-linked to the VA1 promoter. The lower-band form has a polypeptide structure similar to that of the upper-band form except for the absence or modification of the 110-kDa subunit. Direct assays show that the lower-band form is indeed transcriptionally inactive at all stages of purification, even when assayed with an unfractionated, heat-treated nuclear extract as a complementation system. This inactivity does not result from altered DNA-binding properties; instead, we suggest that the alteration of one of the subunits of TFIIIC renders it unable to interact productively with a downstream component of the transcription complex.
authors.affiliation: Laboratory of Biochemistry and Molecular Biology, Rockefeller University, New York, New York 10021.
authors.forename: R
authors.initials: R
authors.lastname: Kovelman
authors.forename: R G
authors.initials: RG
authors.lastname: Roeder
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D002467:Cell Nucleus / Q000378:metabolism*; D002482:Cellulose / Q000031:analogs & derivatives; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004247:DNA; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D015636:Magnesium Chloride / Q000494:pharmacology; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D011189:Potassium Chloride / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D012320:RNA Polymerase III / Q000302:isolation & purification / Q000378:metabolism; D016385:TATA Box; D035361:Transcription Factor TFIIIB; D014157:Transcription Factors / Q000302:isolation & purification* / Q000378:metabolism*; D019474:Transcription Factors, TFIII; D014158:Transcription, Genetic; D000098832:Transcription Factor TFIIIC
chemical_list: C033190:DNA-cellulose; D009838:Oligodeoxyribonucleotides; D035361:Transcription Factor TFIIIB; D014157:Transcription Factors; D019474:Transcription Factors, TFIII; D000098832:Transcription Factor TFIIIC; D015636:Magnesium Chloride; D011189:Potassium Chloride; D002482:Cellulose; D004247:DNA; D012320:RNA Polymerase III