Unique TATA‐binding protein‐containing complexes and cofactors involved in transcription by RNA polymerases II and III

• Published in: The EMBO journal Vol. 12; no. 7; pp. 2749 - 2762
• Main Authors: Chiang, C.M., Ge, H., Wang, Z., Hoffmann, A., Roeder, R.G.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group 01.07.1993
• Subjects: adenovirus; Adenovirus E1B Proteins - genetics; Adenovirus E4 Proteins - genetics; Base Sequence; Biological and medical sciences; Cloning, Molecular; DNA-Binding Proteins - genetics; DNA-Binding Proteins - immunology; DNA-Binding Proteins - metabolism; Epitopes; Fundamental and applied biological sciences. Psychology; HeLa Cells; HIV - genetics; human immunodeficiency virus; Humans; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Mutation; Oligodeoxyribonucleotides; Promoter Regions, Genetic; RNA Polymerase II - metabolism; RNA Polymerase III - metabolism; TATA Box; TATA-Box Binding Protein; Transcription Factor TFIID; Transcription Factor TFIIIB; Transcription Factors - chemistry; Transcription Factors - genetics; Transcription Factors - immunology; Transcription Factors - metabolism; Transcription, Genetic; Transcription. Transcription factor. Splicing. Rna processing; Viral Proteins; Human; Transcription; Enzyme; Transcription promoter; RNA polymerase 3; RNA polymerase 2; Trans acting regulatory factor; Molecular complex; Transcription factor TFIID; In vitro; Hela cell line; Transcription factor TFIIIB
• ISSN: 0261-4189; 1460-2075
• DOI: 10.1002/j.1460-2075.1993.tb05936.x

Two multisubunit complexes containing the TATA‐binding protein (TBP) were isolated from HeLa cells constitutively expressing the FLAG epitope‐tagged TBP using antibody affinity and peptide elution methods. One of the complexes (f:TFIID), isolated from the P11 0.85 M KCl fraction, contains at least 13 specific TBP‐associated factors (TAFs) and can mediate activator‐dependent transcription by RNA polymerase II. Importantly, activator function through the highly purified f:TFIID complex still requires a general cofactor fraction containing upstream factor stimulatory activity (USA). As previously observed with partially purified activator‐competent natural TFIID, f:TFIID generates extended TATA‐dependent footprints on the intrinsically strong adenovirus major late promoter (MLP) but only restricted footprints on the weak adenovirus E1b and E4 and HIV (core) promoters. Along with previous demonstrations of activator‐induced downstream TFIID interactions on the E4 promoter, these results argue for a relationship between downstream interactions and overall promoter strength. Initiator‐like sequences appear not to be essential for downstream interactions since they have no effect on downstream MLP interactions when mutated, do not effect downstream interactions on the HIV promoter and are not present on the inducible E4 promoter. The other multisubunit complex (f:TFIIIB), isolated from the P11 0.30 M KCl fraction, contains four specific TAFs and can substitute for one of the fractions (TFIIIB) required for RNA polymerase III (pol III) transcription. Neither f:TFIID nor TBP could substitute for this pol III TBP‐containing fraction. This plus the fact that f:TFIIIB failed to generate a footprint on the MLP underscores the importance of TAFs in determining promoter specificity by different RNA polymerases.