How Transcriptional Activators Bind Target Proteins

Hermann, Stefan; Berndt, Kurt D.; Wright, Anthony P. H.

doi:10.1074/jbc.m103793200

Cited by 57 publications

(62 citation statements)

References 66 publications

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“…The docking results show that D443 and D445 mostly make hydrogen bonds to K100′ and K77/K79 of PC4, respectively, residues that are important for the interaction (Jonker et al, manuscript in preparation). It is suggested that initial binding occurs by electrostatic interactions, resulting in an unstable complex, whereas it slowly converts into a more stable and defined complex by specific contacts between the TAD and its target (63). In this respect, specific hydrophobic residues may be important.…”

Section: Discussionmentioning

confidence: 99%

Structural Properties of the Promiscuous VP16 Activation Domain

et al. 2004

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Herpes simplex virion protein 16 (VP16) contains two strong activation regions that can independently and cooperatively activate transcription in vivo. We have identified the regions and residues involved in the interaction with the human transcriptional coactivator positive cofactor 4 (PC4) and the general transcription factor TFIIB. NMR and biochemical experiments revealed that both VP16 activation regions are required for the interaction and undergo a conformational transition from random coil to R-helix upon binding to its target PC4. The interaction is strongly electrostatically driven and the binding to PC4 is enhanced by the presence of its amino-terminal domain. We propose models for binding of VP16 to the core domains of PC4 and TFIIB that are based on two independent docking approaches using NMR chemical shift changes observed in titration experiments. The models are consistent with results from site-directed mutagenesis and provide an explanation for the contribution of both acidic and hydrophobic residues for transcriptional activation by VP16. Both intrinsically unstructured activation domains are attracted to their interaction partner by electrostatic interactions, and adopt an R-helical conformation around the important hydrophobic residues. The models showed multiple distinct binding surfaces upon interaction with various partners, providing an explanation for the promiscuous properties, cooperativity, and the high activity of this activation domain.Eukaryotic gene transcription by RNA polymerase II requires the assembly of many proteins on the promoter region (1-3). The rate of transcription is enhanced by transcriptional activators, through recruitment of chromatin remodeling enzymes and through facilitating the assembly of the preinitiation complex (PIC) 1 (4, 5). One of the best characterized activators is the herpes simplex virion protein 16 (VP16), also known as Vmw65, ICP25, or R-TIF (6, 7). The carboxy-terminal region (residues 410-490) of this protein contains two potent transcription activation domains (TADs) (8, 9) that can target many proteins of the RNA polymerase II transcription machinery, such as TBP (10), TFIIA (11,12), TFIIB (13), the RAP74 subunit of TFIIF (14), the p62 component of TFIIH (15), the TBP-associatedfactors hTAF II 31 (16), dTAF II 40 (17), hTAF II 32 (18), the human cofactor PC4 (19,20), CBP (21,22), and p300 (23,24). The two functional regions in this acidic VP16 activation domain (VP16ad) are independently able to activate transcription in vivo via distinct pathways (8,17,22,(25)(26)(27). Both subdomains, VP16ad/n (412-453) and VP16ad/c (454-490), have been extensively studied by mutational analysis, indicating key roles for specific hydrophobic and acidic residues (26-29). The TADs of transcription factors often lack a folded structure under physiological conditions †

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Section: Discussionmentioning

confidence: 99%

Structural Properties of the Promiscuous VP16 Activation Domain

et al. 2004

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“…Current models of the function of transcriptional activator require specific charge and hydrophobic interactions to strengthen binding between conformationally flexible TADs and partner proteins (Hermann et al 2001, Razeto et al 2004, Zor et al 2004. The lack of constraint for sidechain length (Fig.…”

Section: Discussionmentioning

confidence: 99%

Repression of the prolactin promoter: a functional consequence of the heterodimerization between Pit-1 and Pit-1 β

Sporici

Hodskins²,

Locasto³

et al. 2005

Journal of Molecular Endocrinology

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The POU-homeodomain transcription factor Pit-1 is required for the differentiation of the anterior pituitary cells and the expression of their hormone products. Pit-1 , an alternate splicing isoform, has diametrically different outcomes when it is expressed in different cell types. Pit-1 acts as a transcriptional repressor of prolactin (PRL) and growth hormone genes in pituitary cells, and as a transcriptional activator in non-pituitary cells. In order to explore these differences, we: (1) identified the transcriptional cofactors necessary for reconstitution of repression in non-pituitary cells; (2) tested the effect of the -domain on heterodimerization with Pit-1 and physical interaction with the co-activator CREB binding protein (CBP); and (3) determined the -domain sidechain chemistry requirements for repression. Co-expression of both Pit-1 isoforms reconstituted the repression of the PRL promoter in non-pituitary cells. The -domain allowed heterodimerization with Pit-1 but blocked physical interaction with CBP, and specific chemical properties of the -domain beyond hydrophobicity were dispensable. These data strongly suggest that Pit-1 represses hormone gene expression by heterodimerizing with Pit-1 and interfering with the assembly of the Pit-1-CBP complex required for PRL promoter activity in pituitary cells.

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“…GST and GST fusion proteins were dialyzed against phosphate-buffered saline. Yeast TBP was purified and treated as described previously (8). All proteins were frozen immediately following purification and subsequently stored at Ϫ70°C until use.…”

Section: Methodsmentioning

confidence: 99%

“…subtraction of background binding to the affinity tag (GST), as well as adjustment for flow cell-specific bulk effects by subtraction of buffer injection). Curve fitting was performed using BIAevaluation 3.0.2 software (BIA-CORE) and a sequential two-step model as previously described (8).…”

Section: Methodsmentioning

confidence: 99%

“…In addition, mutations, causing substitutions of hydrophobic residues that might be expected to affect folding of the glucocorticoid receptor tau1 activation domain have been shown to cause changes in the activity of the intact receptor protein (7). We have previously reported that the activation domain from the cancer-related c-Myc protein interacts with the TATA-binding protein (TBP) in two steps, such that folding of the c-Myc activation domain is coupled to its interaction with the surface of TBP (8). Coupled binding and folding could explain how activators are able to interact specifically with a large set of structurally different targets in the transcriptional machinery and recruit them to promoters.…”

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confidence: 99%

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Mechanism of Transcription Factor Recruitment by Acidic Activators

Ferreira

Hermann

Prochasson

et al. 2005

Journal of Biological Chemistry

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Many transcriptional activators are intrinsically unstructured yet display unique, defined conformations when bound to target proteins. Target-induced folding provides a mechanism by which activators could form specific interactions with an array of structurally unrelated target proteins. Evidence for such a binding mechanism has been reported previously in the context of the interaction between the cancer-related c-Myc protein and the TATA-binding protein, which can be modeled as a two-step process in which a rapidly forming, low affinity complex slowly converts to a more stable form, consistent with a coupled binding and folding reaction. To test the generality of the target-induced folding model, we investigated the binding of two widely studied acidic activators, Gal4 and VP16, to a set of target proteins, including TATA-binding protein and the Swi1 and Snf5 subunits of the Swi/Snf chromatin remodeling complex. Using surface plasmon resonance, we show that these activator-target combinations also display bi-phasic kinetics suggesting two distinct steps. A fast initial binding phase that is inhibited by high ionic strength is followed by a slow phase that is favored by increased temperature. In all cases, overall affinity increases with temperature and, in most cases, with increased ionic strength. These results are consistent with a general mechanism for recruitment of transcriptional components to promoters by naturally occurring acidic activators, by which the initial contact is mediated predominantly through electrostatic interactions, whereas subsequent target-induced folding of the activator results in a stable complex.

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How Transcriptional Activators Bind Target Proteins

Cited by 57 publications

References 66 publications

Structural Properties of the Promiscuous VP16 Activation Domain

Structural Properties of the Promiscuous VP16 Activation Domain

Repression of the prolactin promoter: a functional consequence of the heterodimerization between Pit-1 and Pit-1 β

Mechanism of Transcription Factor Recruitment by Acidic Activators

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