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

Cheng Ming Chiang, Hui Ge, Zhengxin Wang, Alexander Hoffmann, Robert G. Roeder

Research output: Contribution to journalArticle

167 Scopus citations

Abstract

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:TFIID), isolated from the P11 0.30 M KCl fraction, contains four specific TAFs and can substitute for one of the fractions (TFIIB) 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.

Original languageEnglish (US)
Pages (from-to)2749-2762
Number of pages14
JournalEMBO Journal
Volume12
Issue number7
DOIs
StatePublished - Jan 1 1993

Keywords

  • Epitope-tagged TBP
  • TAF
  • TFIIB
  • TFIID
  • Transcription

ASJC Scopus subject areas

  • Neuroscience(all)
  • Molecular Biology
  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)

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