Purification of a yeast TATA box-binding protein that exhibits human transcription factor IID activity.

Horikoshi M, Wang CK, Fujii H, Cromlish JA, Weil PA, Roeder RG
Proc Natl Acad Sci U S A. 1989 86 (13): 4843-7

PMID: 2662184 · PMCID: PMC297511 · DOI:10.1073/pnas.86.13.4843

By a series of conventional chromatographic procedures we have purified from whole-cell extracts of Saccharomyces cerevisiae yeast transcription factor IID (TFIID), which functionally substitutes for human TFIID in a complementation assay comprised of the adenovirus type 2 major late promoter and HeLa cell-derived RNA polymerase II, transcription factors IIA, IIB, and IIE. Similar to its human counterpart, yeast TFIID also exhibited specific binding to the adenovirus type 2 major late promoter TATA element, as shown by both DNase I footprinting and gel mobility shift assays. NaDodSO4/PAGE analyses showed that a 27-kDa polypeptide coeluted with TFIID complementing activity through each chromatographic step. In agreement with this result and also suggesting that the native protein is a monomer, gel-filtration experiments indicated a molecular mass of 28 kDa for TFIID under nondenaturing conditions. That the 27-kDa polypeptide represented TFIID was further demonstrated by the ability of an HPLC-purified protein to bind specifically after renaturation to the adenovirus type 2 major late promoter TATA sequence.

MeSH Terms (15)

Antigens, Neoplasm Base Sequence Chromatography, Affinity Chromatography, Gel Fagopyrum HeLa Cells Histocompatibility Antigens Humans Kinetics Molecular Weight Promoter Regions, Genetic Saccharomyces cerevisiae Transcription, Genetic Transcription Factors Transcription Factor TFIID

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