Purification and characterization of two forms of human transcription factor IIIC.
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.
Kovelman
R
R
Laboratory of Biochemistry and Molecular Biology, Rockefeller University, New York, New York 10021.
Roeder
R G
RG
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
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