TATA-Binding Protein Recognition and Bending of a Consensus Promoter Are Protein Species Dependent

Whittington, JoDell E.; Delgadillo, Roberto F.; Attebury, Torrissa J.; Parkhurst, Laura K.; Daugherty, Margaret A.; Parkhurst, Lawrence J.

doi:10.1021/bi800139w

Cited by 22 publications

(55 citation statements)

References 46 publications

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“…Our model suggests TBP shows little discrimination between sequences during association and sequence specificity is defined by the sequence's kinetic stability. This agrees with the observation that the first phase of TBP binding (in the three-step model) is independent of TBP concentration, as would be expected in our model (42). …”

Section: Discussionsupporting

confidence: 93%

“…However, to elucidate the precise conformational states for each DNA, more powerful techniques are needed. For example, previous studies suggested that two or more different intermediate states are present during binding of TBP to a minimal TATA-DNA (13,16), while more recent spFRET studies on human TBP indicate a two-state system (37,42). To test whether flanking DNA influences the formation of any intermediate states, we used single-pair FRET (spFRET) spectroscopy (43) to resolve the specific DNA conformations induced by TBP binding.…”

Section: Resultsmentioning

confidence: 99%

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The conformational state of the nucleosome entry–exit site modulates TATA box-specific TBP binding

et al. 2014

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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.

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Section: Discussionsupporting

confidence: 93%

Section: Resultsmentioning

confidence: 99%

The conformational state of the nucleosome entry–exit site modulates TATA box-specific TBP binding

et al. 2014

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“…Additional controls to correct for spectral overlap must be performed (see below). While FRET has been utilized to measure high-affinity interactions inferred from intramolecular distance changes (9,23,24), it has not been widely used for measuring intermolecular interactions between labeled components. Several recent descriptions for quantitative intermolecular FRET affinity measurements have been published; however these experiments rely upon high probe concentrations and are not amenable to high-affinity interactions (11,25,26).…”

Section: Resultsmentioning

confidence: 99%

Fluorescence strategies for high-throughput quantification of protein interactions

Hieb

D’Arcy

Krämer

et al. 2011

Nucleic Acids Research

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Advances in high-throughput characterization of protein networks in vivo have resulted in large databases of unexplored protein interactions that occur during normal cell function. Their further characterization requires quantitative experimental strategies that are easy to implement in laboratories without specialized equipment. We have overcome many of the previous limitations to thermodynamic quantification of protein interactions, by developing a series of in-solution fluorescence-based strategies. These methods have high sensitivity, a broad dynamic range, and can be performed in a high-throughput manner. In three case studies we demonstrate how fluorescence (de)quenching and fluorescence resonance energy transfer can be used to quantitatively probe various high-affinity protein–DNA and protein–protein interactions. We applied these methods to describe the preference of linker histone H1 for nucleosomes over DNA, the ionic dependence of the DNA repair enzyme PARP1 in DNA binding, and the interaction between the histone chaperone Nap1 and the histone H2A–H2B heterodimer.

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“…We monitored the dynamics of the TBP–DNA TATA interaction on the single-molecule level using a FRET signal as readout that allowed us to follow the TBP-induced bending of the promoter DNA (27) (Figure 1C). As TBP binding is inseparably connected to DNA bending, this assay provides information about both complex formation and DNA bending (22). In order to ensure biological relevant temperatures for the archaeal systems, we equipped our single-molecule total internal reflection fluorescence (TIRF) setup with a heating module that allowed temperature-controlled measurements up to 60°C.…”

Section: Introductionmentioning

confidence: 99%

Eukaryotic and archaeal TBP and TFB/TF(II)B follow different promoter DNA bending pathways

Gietl

Holzmeister

Blombach

et al. 2014

Nucleic Acids Research

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During transcription initiation, the promoter DNA is recognized and bent by the basal transcription factor TATA-binding protein (TBP). Subsequent association of transcription factor B (TFB) with the TBP–DNA complex is followed by the recruitment of the ribonucleic acid polymerase resulting in the formation of the pre-initiation complex. TBP and TFB/TF(II)B are highly conserved in structure and function among the eukaryotic-archaeal domain but intriguingly have to operate under vastly different conditions. Employing single-pair fluorescence resonance energy transfer, we monitored DNA bending by eukaryotic and archaeal TBPs in the absence and presence of TFB in real-time. We observed that the lifetime of the TBP–DNA interaction differs significantly between the archaeal and eukaryotic system. We show that the eukaryotic DNA-TBP interaction is characterized by a linear, stepwise bending mechanism with an intermediate state distinguished by a distinct bending angle. TF(II)B specifically stabilizes the fully bent TBP–promoter DNA complex and we identify this step as a regulatory checkpoint. In contrast, the archaeal TBP–DNA interaction is extremely dynamic and TBP from the archaeal organism Sulfolobus acidocaldarius strictly requires TFB for DNA bending. Thus, we demonstrate that transcription initiation follows diverse pathways on the way to the formation of the pre-initiation complex.

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TATA-Binding Protein Recognition and Bending of a Consensus Promoter Are Protein Species Dependent

Cited by 22 publications

References 46 publications

The conformational state of the nucleosome entry–exit site modulates TATA box-specific TBP binding

The conformational state of the nucleosome entry–exit site modulates TATA box-specific TBP binding

Fluorescence strategies for high-throughput quantification of protein interactions

Eukaryotic and archaeal TBP and TFB/TF(II)B follow different promoter DNA bending pathways

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