Dynamic interplay of TFIIA, TBP and TATA DNA

Christian A. Weideman, Robert C. Netter, Lawrence R. Benjamin, John J. McAllister, Lumelle A. Schmiedekamp, Robert A. Coleman, B. Franklin Pugh

Research output: Contribution to journalArticle

47 Scopus citations

Abstract

The TATA binding protein (TBP) binds to the -30 region of eukaryotic and archaea promoters, where it assembles a transcription complex. For those genes transcribed by RNA polymerase II, transcription factor TFIIA binds TBP and positively regulates its activity, including enhancing TBP/ TATA interactions. Since little is known about the dynamic interplay among TFIIA, TBP and DNA, we set out to examine the stability of these interactions. Using the nitrocellulose filter binding assay, the k(off) of recombinant human TBP from TATA and non-specific DNA was determined to be 5.5(± 0.1) x 10-5 s-1 (t(1/2) = 210 minutes) and 5.8(± 0.1) x 10-4 s-1 (t(1/2) = 20 minutes), respectively. TFIIA/TBP complexes, containing either HeLa-derived or recombinant human TFIIA, possessed a nearly tenfold lower k(off) when bound to TATA. Interactions of TFIIA with DNA upstream of the TATA box did not appear to play a major role in stabilizing TBP/TATA interactions. Instead, the upstream DNA contacts appeared to be important for stabilizing the association of TFIIA with the TBP/TATA complex as measured in electrophoretic mobility shift assays: k(off) of TFIIA decreased from 1.4(± 0.1)x 10-3 s-1 (t(1/2) = eight minutes) to 2.4(± 0.2) x 10-4 s-1 (t(1/2) = 49 minutes) when upstream DNA contacts were allowed. The stability of TFIIA/TBP interactions was measured using a rapid 'pull-down' assay, which employed-nickel agarose and polyhistidine-tagged TFIIA. In the absence of DNA, TFIIA dissociated from TBP with a k(off) = 4.9(± 0.6) x 10-3 s-1 (t(1/2) = 2.4 minutes), which varied with solution conditions.

Original languageEnglish (US)
Pages (from-to)61-75
Number of pages15
JournalJournal of Molecular Biology
Volume271
Issue number1
DOIs
StatePublished - Aug 8 1997
Externally publishedYes

Keywords

  • Kinetics
  • Protein-DNA interactions
  • TATA box
  • TBP
  • TFIIA

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

Fingerprint Dive into the research topics of 'Dynamic interplay of TFIIA, TBP and TATA DNA'. Together they form a unique fingerprint.

  • Cite this

    Weideman, C. A., Netter, R. C., Benjamin, L. R., McAllister, J. J., Schmiedekamp, L. A., Coleman, R. A., & Pugh, B. F. (1997). Dynamic interplay of TFIIA, TBP and TATA DNA. Journal of Molecular Biology, 271(1), 61-75. https://doi.org/10.1006/jmbi.1997.1152