title: Factors involved in specific transcription by mammalian RNA polymerase II: purification, genetic specificity, and TATA box-promoter interactions of TFIID.
abstract: Selective and accurate transcription of purified genes by RNA polymerase II requires multiple factors. The factor designated TFIID was purified extensively from HeLa cell nuclear extracts by using a simple and novel complementation assay. Thus, TFIID was preferentially inactivated by mild heat treatment of a nuclear extract, and supplementation of the heat-treated extract with TFIID-containing fractions restored adenovirus major late (ML) promoter-dependent transcription. By using this assay, TFIID was purified approximately 300-fold by conventional chromatographic methods. The most purified TFIID fraction was demonstrated to be required for transcription of a number of other cellular and viral class II genes. This factor showed specific interactions with both the adenovirus ML promoter and a human heat shock 70 (hsp-70) promoter. On the ML promoter, the DNase I-protected region extended from around position -40 to position +35, although some discontinuities (and associated hypersensitive sites) were apparent near the initiation site and near position +27; the upstream and downstream boundaries of the TFIID-binding site were also confirmed by exonuclease III digestion experiments. In contrast to these results, the DNase I-protected regions on the human hsp-70 promoter were confined to a smaller area that extended from positions -35 to -19. DNase I hypersensitive sites were observed in both the adenovirus ML and hsp-70 promoters, most notably in the region at position -47. These results indicate either that there are different forms of TFIID or that a single TFIID can interact differently with distinct promoters.
authors.affiliation: Laboratory of Biochemistry and Molecular Biology, Rockefeller University, New York, New York 10021.
authors.forename: N
authors.initials: N
authors.lastname: Nakajima
authors.forename: M
authors.initials: M
authors.lastname: Horikoshi
authors.forename: R G
authors.initials: RG
authors.lastname: Roeder
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D001483:Base Sequence; D006367:HeLa Cells; D006360:Heat-Shock Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012319:RNA Polymerase II / Q000378:metabolism*; D012045:Regulatory Sequences, Nucleic Acid; D035362:Transcription Factor TFIID; D014157:Transcription Factors / Q000302:isolation & purification* / Q000502:physiology*; D014158:Transcription, Genetic
chemical_list: D006360:Heat-Shock Proteins; D035362:Transcription Factor TFIID; D014157:Transcription Factors; D012319:RNA Polymerase II