The conformational state of the nucleosome entry-exit site modulates TATA box-specific TBP binding.
The TATA binding protein (TBP) is a critical transcription factor used for nucleating assembly of the RNA polymerase II machinery. TBP binds TATA box elements with high affinity and kinetic stability and in vivo is correlated with high levels of transcription activation. However, since most promoters use less stable TATA-less or TATA-like elements, while also competing with nucleosome occupancy, further mechanistic insight into TBP's DNA binding properties and ability to access chromatin is needed. Using bulk and single-molecule FRET, we find that TBP binds a minimal consensus TATA box as a two-state equilibrium process, showing no evidence for intermediate states. However, upon addition of flanking DNA sequence, we observe non-specific cooperative binding to multiple DNA sites that compete for TATA-box specificity. Thus, we conclude that TBP binding is defined by a branched pathway, wherein TBP initially binds with little sequence specificity and is thermodynamically positioned by its kinetic stability to the TATA box. Furthermore, we observed the real-time access of TBP binding to TATA box DNA located within the DNA entry-exit site of the nucleosome. From these data, we determined salt-dependent changes in the nucleosome conformation regulate TBP's access to the TATA box, where access is highly constrained under physiological conditions, but is alleviated by histone acetylation and TFIIA.
Division Biophysics of Macromolecules, German Cancer Research Center, D-69120 Heidelberg, Germany.
Aaron R
AR
Hieb
Division Biophysics of Macromolecules, German Cancer Research Center, D-69120 Heidelberg, Germany.
Alexander
A
Gansen
Division Biophysics of Macromolecules, German Cancer Research Center, D-69120 Heidelberg, Germany.
Vera
V
Böhm
Division Biophysics of Macromolecules, German Cancer Research Center, D-69120 Heidelberg, Germany jl@dkfz.de.
Jörg
J
Langowski
D000107:Acetylation; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D006657:Histones / Q000378:metabolism; D009690:Nucleic Acid Conformation; D009707:Nucleosomes / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D016385:TATA Box; D035181:TATA-Box Binding Protein / Q000378:metabolism*; D035561:Transcription Factor TFIIA / Q000378:metabolism
D006657:Histones; D009707:Nucleosomes; D035181:TATA-Box Binding Protein; D035561:Transcription Factor TFIIA; D004247:DNA