Kinetic Preference for Oriented DNA Binding by the Yeast TATA-Binding Protein TBP

Liu, Yichin; Schepartz, Alanna

doi:10.1021/bi0019794

Cited by 13 publications

(10 citation statements)

References 48 publications

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“…preferentially biased differs from that derived from similar studies using either 1,10-phenanthroline or fluorescence resonance energy transfer to probe the orientation of TATA boxbound TBP and TBPc, respectively, that demonstrated only a weak bias for TBP binding in the correct orientation (8,48). There is no obvious reason for the differences observed between the studies.…”

Section: Discussioncontrasting

confidence: 82%

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Function and Structural Organization of Mot1 Bound to a Natural Target Promoter

Sprouse

Shcherbakova

Cheng

et al. 2008

Journal of Biological Chemistry

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Mot1 is an essential, conserved TATA-binding protein (TBP)-associated factor in Saccharomyces cerevisiae and a member of the Snf2/Swi2 ATPase family. Mot1 uses ATP hydrolysis to displace TBP from DNA, an activity that can be readily reconciled with its global role in gene repression. Less well understood is how Mot1 directly activates gene expression. It has been suggested that Mot1-mediated activation can occur by displacement of inactive TBP-containing complexes from promoters, thereby permitting assembly of functional transcription complexes. Mot1 may also activate transcription by other mechanisms that have not yet been defined. A gap in our understanding has been the absence of biochemical information related to the activity of Mot1 on natural target genes. Using URA1 as a model Mot1-activated promoter, we show striking differences in the way that both TBP and Mot1 interact with DNA compared with other model DNA substrates analyzed previously. These differences are due at least in part to the propensity of TBP alone to bind to the URA1 promoter in the wrong orientation to direct appropriate assembly of the URA1 preinitiation complex. The results suggest that Mot1-mediated activation of URA1 transcription involves at least two steps, one of which is the removal of TBP bound to the promoter in the opposite orientation required for URA1 transcription.The TATA-binding protein (TBP) 2 is a central component of the RNA polymerase II (pol II) preinitiation complex (PIC) (1-3). Transcription can be regulated during many different steps in PIC assembly, but the regulation of TBP binding to promoters is a fundamentally important step exploited for regulation of a large number of genes (4, 5). TBP is a saddle-shaped protein that interacts with DNA as a monomer (6, 7). Because TBP can straddle DNA in either of two directions, its binding polarity determines the orientation of the assembled PIC and hence the direction in which transcription initiates (8). TBP recruitment to promoters is regulated by chromatin, activators, and co-activators that contact TBP directly, and general factors that modulate TBP binding and activity (1, 2, 4, 9 -11).NC2 and Mot1 are essential, conserved regulators of TBP function that cooperate to regulate gene expression on a global scale (12)(13)(14). NC2, a heterodimer of the Bur6 and Ydr1 subunits in yeast (15), interacts with the TBP-DNA complex and can block subsequent steps in PIC assembly (16). NC2 is also a direct activator of gene expression (17) that is found at promoters in vivo in proportion to the level of transcription (14). How this apparent negative regulator of transcription functions as an activator is likely related to the recent observation that NC2 binding can allow TBP to relocalize along the DNA contour (18). Such mobilization of TBP may be critical for clearance of TBP from inactive but high affinity sites, and may facilitate selection of appropriate TATA sequences among a collection of binding sites along accessible promoter DNA. Mot1 is a member of the Snf2/Swi2 ATPa...

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Section: Discussioncontrasting

confidence: 82%

“…No DNA reactivity was observed adjacent to Lys-97 or in-phase on the downstream DNA. We consider this interpretation under "Discussion" in relation to published TBP-DNA-directed cleavage patterns (8,48).…”

Section: Dna Downstream Of Tata Is Required For Mot1mentioning

confidence: 99%

Function and Structural Organization of Mot1 Bound to a Natural Target Promoter

Sprouse

Shcherbakova

Cheng

et al. 2008

Journal of Biological Chemistry

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“…The interactions of Saccharomyces cerevisiae TBP with promoters bearing two consensus sequences, adenovirus major late (TATAAAAG, AdMLP) and E4 (TATATATA), have been well characterized via extensive biochemical (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22), crystallographic (23)(24)(25)(26)(27)(28), and molecular dynamics (29 -33) studies. Both promoters bind to TBP at rates significantly slower than diffusion limited (12,13,(15)(16)(17)(18)(19)(20), forming tightly bound complexes with similar co-crystal structures (23)(24)(25)(26)(27) and comparable DNA geometries in solution (34).…”

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confidence: 99%

Comparison of TATA-binding Protein Recognition of a Variant and Consensus DNA Promoters

Powell

Parkhurst

2002

Journal of Biological Chemistry

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Assembly of transcription pre-initiation complexes proceeds from the initial complex formed between "TATA" bearing promoter DNA and the TATA-binding protein (TBP). Our laboratory has been investigating the relationships among TATA sequence, TBP⅐TATA solution structure, recognition mechanisms, and transcription efficiency. TBP⅐TATA interactions have been modeled by global analysis of detailed kinetic and thermodynamic data obtained using fluorimetric and fluorometric techniques in conjunction with fluorescence resonance energy transfer. We have reported recently that TBP recognition of two consensus promoters, adenovirus major late (AdMLP: TATAAAAG) and E4 (TATATATA), is well described by a linear two-intermediate mechanism with simultaneous DNA binding and bending. Similar DNA geometries and high transcription efficiencies characterize these TBP⅐TATA complexes. Here we show that, in contrast to the consensus sequences, TBP recognition of a variant sequence (C7: TATAAACG) is described by a three-step model with two branching pathways. One pathway proceeds through an intermediate having severely bent DNA, reminiscent of the consensus interactions, with the other branch yielding a unique conformer with shallowly bent DNA. The resulting TBP⅐C7 complex has a dramatically different solution conformation than for TBP⅐DNA CONSENSUS and is correlated with diminished relative transcription activity. The temperature dependence of the TBP⅐C7 helical bend is postulated to derive from population shifts between the conformers with slightly and severely bent DNA.For eukaryotic class II genes, the binary complex formed between the TATA-binding protein and a conserved promoter site, the TATA box, provides the foundation upon which the transcription pre-initiation complex is assembled (1-7). TBP 1 binds productively to consensus (TATA(a/t)A(a/t)N) and diverse variant TATA sequences (8, 9), yielding relative transcription efficiencies ranging from Ͻ1 to 172 (9, 10).The interactions of Saccharomyces cerevisiae TBP with promoters bearing two consensus sequences, adenovirus major late (TATAAAAG, AdMLP) and E4 (TATATATA), have been well characterized via extensive biochemical (11-22), crystallographic (23-28), and molecular dynamics (29 -33) studies. Both promoters bind to TBP at rates significantly slower than diffusion limited (12,13,(15)(16)(17)(18)(19)(20), forming tightly bound complexes with similar co-crystal structures (23-27) and comparable DNA geometries in solution (34). Additionally, these two sequences yield high relative transcription activities (9, 10).Our laboratory has been studying the detailed recognition mechanism of DNA promoters by S. cerevisiae TBP (15,16,20) and the solution geometries of the resulting TBP⅐TATA complexes (34, 35). Both lines of investigation utilize dye-labeled TATA-bearing oligomers and steady-state, stopped-flow, and time-resolved fluorescence techniques in conjunction with fluorescence resonance energy transfer (FRET). Global analysis of extensive real-time kinetic and thermodynamic data sets...

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“…1). Although it is only observed in one orientation in x-ray structures, Schepartz and coworkers have shown that TBP can bind to the TATA box in two orientations [13][14][15] . The direction of Polymerase II translation along the DNA is determined by the orientation of TBP 16 on the TATA box.…”

Section: Resultsmentioning

confidence: 99%

Dynamics of TBP binding to the TATA box

et al. 2008

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Gene expression is highly controlled and regulated in living cells. One of the first steps in gene transcription is recognition of the promoter site by the TATA box Binding Protein (TBP). TBP recruits other transcriptions factors and eventually the RNA polymerase II to transcribe the DNA in mRNA. We developed a single pair Förster Resonance Energy Transfer (spFRET) assay to investigate the mechanism of gene regulation. Here, we apply this assay to investigate the initial binding process of TBP to the adenovirus major late (AdML) promoter site. From the spFRET measurements, we were able to identify two conformations of the TBP-DNA complex that correspond to TBP bound in the correct and the opposite orientation. Increased incubation times or the presence of the transcription factor TFIIA improved the alignment of TBP on the promoter site. Binding of TBP to the TATA box shows a rich dynamics with abrupt transitions between multiple FRET states. A frame-wise histogram analysis revealed the presence of at least six discrete states, showing that TBP binding is more complicated than previously thought. Hence, the spFRET assay is very sensitive to the conformation of the TBP-DNA complex and is very promising tool for investigating the pathway of TBP binding in detail.

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Kinetic Preference for Oriented DNA Binding by the Yeast TATA-Binding Protein TBP

Cited by 13 publications

References 48 publications

Function and Structural Organization of Mot1 Bound to a Natural Target Promoter

Function and Structural Organization of Mot1 Bound to a Natural Target Promoter

Comparison of TATA-binding Protein Recognition of a Variant and Consensus DNA Promoters

Dynamics of TBP binding to the TATA box

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