TFIIA changes the conformation of the DNA in TBP/TATA complexes and increases their kinetic stability.
Eukaryotic mRNA transcription by RNA polymerase II is a highly regulated complex reaction involving numerous proteins. In order to control tissue and promoter specific gene expression, transcription factors must work in concert with each other and with the promoter DNA to form the proper architecture to activate the gene of interest. The TATA binding protein (TBP) binds to TATA boxes in core promoters and bends the TATA DNA. We have used quantitative solution fluorescence resonance energy transfer (FRET) and gel-based FRET (gelFRET) to determine the effect of TFIIA on the conformation of the DNA in TBP/TATA complexes and on the kinetic stability of these complexes. Our results indicate that human TFIIA decreases the angle to which human TBP bends consensus TATA DNA from 104 degrees to 80 degrees when calculated using a two-kink model. The kinetic stability of TBP/TATA complexes was greatly reduced by increasing the KCl concentration from 50 mM to 140 mM, which is more physiologically relevant. TFIIA significantly enhanced the kinetic stability of TBP/TATA complexes, thereby attenuating the effect of higher salt concentrations. We also found that TBP bent non-consensus TATA DNA to a lesser degree than consensus TATA DNA and complexes between TBP and a non-consensus TATA box were kinetically unstable even at 50 mM KCl. Interestingly, TFIIA increased the calculated bend angle and kinetic stability of complexes on a non-consensus TATA box, making them similar to those on a consensus TATA box. Our data show that TFIIA induces a conformational change within the TBP/TATA complex that enhances its stability under both in vitro and physiological salt conditions. Furthermore, we present a refined model for the effect that TFIIA has on DNA conformation that takes into account potential changes in bend angle as well as twist angle.
Department of Chemistry and Biochemistry, University of Colorado at Boulder, 215 UCB, Boulder, CO 80309-0215, USA.
Aaron R
AR
Hieb
Wayne A
WA
Halsey
Meredith D
MD
Betterton
Thomas T
TT
Perkins
Jennifer F
JF
Kugel
James A
JA
Goodrich
D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D009690:Nucleic Acid Conformation; D016385:TATA Box / Q000235:genetics*; D035181:TATA-Box Binding Protein / Q000737:chemistry* / Q000378:metabolism*; D013816:Thermodynamics; D035561:Transcription Factor TFIIA / Q000378:metabolism*
D035181:TATA-Box Binding Protein; D035561:Transcription Factor TFIIA; D004247:DNA