Purification of a yeast TATA box-binding protein that exhibits human transcription factor IID activity.
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.
Horikoshi
M
M
Laboratory of Biochemistry and Molecular Biology, Rockefeller University, New York, NY 10021.
Wang
C K
CK
Fujii
H
H
Cromlish
J A
JA
Weil
P A
PA
Roeder
R G
RG
D000951:Antigens, Neoplasm; D001483:Base Sequence; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D019612:Fagopyrum / Q000276:immunology / Q000378:metabolism*; D006367:HeLa Cells / Q000378:metabolism; D006649:Histocompatibility Antigens; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D011401:Promoter Regions, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D035362:Transcription Factor TFIID; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic
D000951:Antigens, Neoplasm; D006649:Histocompatibility Antigens; D035362:Transcription Factor TFIID; D014157:Transcription Factors; C029237:tumor-associated transplantation antigen