title: Single-molecule fluorescence resonance energy transfer shows uniformity in TATA binding protein-induced DNA bending and heterogeneity in bending kinetics.
abstract: TATA binding protein (TBP) is a key component of the eukaryotic RNA polymerase II transcription machinery that binds to TATA boxes located in the core promoter regions of many genes. Structural and biochemical studies have shown that when TBP binds DNA, it sharply bends the DNA. We used single-molecule fluorescence resonance energy transfer (smFRET) to study DNA bending by human TBP on consensus and mutant TATA boxes in the absence and presence of TFIIA. We found that the state of the bent DNA within populations of TBP-DNA complexes is homogeneous; partially bent intermediates were not observed. In contrast to the results of previous ensemble studies, TBP was found to bend a mutant TATA box to the same extent as the consensus TATA box. Moreover, in the presence of TFIIA, the extent of DNA bending was not significantly changed, although TFIIA did increase the fraction of DNA molecules bound by TBP. Analysis of the kinetics of DNA bending and unbending revealed that on the consensus TATA box two kinetically distinct populations of TBP-DNA complexes exist; however, the bent state of the DNA is the same in the two populations. Our smFRET studies reveal that human TBP bends DNA in a largely uniform manner under a variety of different conditions, which was unexpected given previous ensemble biochemical studies. Our new observations led to us to revise the model for the mechanism of DNA binding by TBP and for how DNA bending is affected by TATA sequence and TFIIA.
authors.affiliation: Department of Chemistry and Biochemistry, University of Colorado, 215 UCB, Boulder, CO 80309-0215, USA.
authors.forename: Rebecca H
authors.initials: RH
authors.lastname: Blair
authors.forename: James A
authors.initials: JA
authors.lastname: Goodrich
authors.forename: Jennifer F
authors.initials: JF
authors.lastname: Kugel
mesh_terms: D004247:DNA / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D007700:Kinetics; D009690:Nucleic Acid Conformation; D016385:TATA Box; D035181:TATA-Box Binding Protein / Q000502:physiology*
chemical_list: D035181:TATA-Box Binding Protein; D004247:DNA