scholarly journals Transcription activator-coactivator specificity is mediated by a large and dynamic fuzzy protein-protein complex

2017 ◽  
Author(s):  
Lisa M. Tuttle ◽  
Derek Pacheco ◽  
Linda Warfield ◽  
Jie Luo ◽  
Jeff Ranish ◽  
...  
Keyword(s):  
Transcription Activation ◽  
Disordered Proteins ◽  
Protein Interface ◽  
Transcription Activator ◽  
Great Flexibility ◽  
Hydrophobic Residues ◽  
Activation Domains ◽  
Binding Domains ◽  
Complex Forms ◽  
Combinatorial Mechanism

SUMMARYTranscription activation domains (ADs) are inherently disordered proteins that often target multiple coactivator complexes, but the specificity of these interactions is not understood. Efficient activation by yeast Gcn4 requires tandem Gcn4 ADs and four activator-binding domains (ABDs) on its target, the Mediator subunit Med15. Multiple ABDs are a common feature of coactivator complexes. We find that the large Gcn4-Med15 complex is heterogeneous, containing nearly all possible AD-ABD interactions. This complex forms using a dynamic fuzzy protein-protein interface where ADs use hydrophobic residues to bind hydrophobic surfaces of the ABDs in multiple orientations. This combinatorial mechanism allows individual interactions of low affinity and specificity to generate a biologically functional, specific, and higher affinity complex despite lacking a defined protein-protein interface. This binding strategy is likely representative of many activators that target multiple coactivators and allows great flexibility in combinations of activators that synergize to regulate genes with variable coactivator requirements.

scholarly journals Transcription Activation Domains of the Yeast Factors Met4 and Ino2: Tandem Activation Domains with Properties Similar to the Yeast Gcn4 Activator

2018 ◽  
Vol 38 (10) ◽  
Author(s):  
Derek Pacheco ◽  
Linda Warfield ◽  
Michelle Brajcich ◽  
Hannah Robbins ◽  
Jie Luo ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Transcription Activation ◽  
Binding Studies ◽  
Eukaryotic Transcription ◽  
Activation Domains ◽  
Conserved Sequence ◽  
Functional Studies ◽  
Intrinsically Disordered ◽  
Binding Domains ◽  
Optimal Function

ABSTRACTEukaryotic transcription activation domains (ADs) are intrinsically disordered polypeptides that typically interact with coactivator complexes, leading to stimulation of transcription initiation, elongation, and chromatin modifications. Here we examined the properties of two strong and conserved yeast ADs: Met4 and Ino2. Both factors have tandem ADs that were identified by conserved sequence and functional studies. While the AD function of both factors depended on hydrophobic residues, Ino2 further required key conserved acidic and polar residues for optimal function. Binding studies showed that the ADs bound multiple Med15 activator-binding domains (ABDs) with similar orders of micromolar affinity and similar but distinct thermodynamic properties. Protein cross-linking data show that no unique complex was formed upon Met4-Med15 binding. Rather, we observed heterogeneous AD-ABD contacts with nearly every possible AD-ABD combination. Many of these properties are similar to those observed with yeast activator Gcn4, which forms a large heterogeneous, dynamic, and fuzzy complex with Med15. We suggest that this molecular behavior is common among eukaryotic activators.

Inhibition of Xbra transcription activation causes defects in mesodermal patterning and reveals autoregulation of Xbra in dorsal mesoderm

Development ◽  
1996 ◽  
Vol 122 (8) ◽  
pp. 2427-2435 ◽  
Author(s):  
F.L. Conlon ◽  
S.G. Sedgwick ◽  
K.M. Weston ◽  
J.C. Smith
Keyword(s):  
Transcription Activation ◽  
Zebrafish Embryos ◽  
Transcription Activator ◽  
Activation Domain ◽  
Activation Domains ◽  
Dorsal Mesoderm ◽  
Transcription Activators ◽  
Carboxy Terminal ◽  
Genetic Mutants ◽  
Axial Development

The Brachyury (T) gene is required for formation of posterior mesoderm and for axial development in both mouse and zebrafish embryos. In this paper, we first show that the Xenopus homologue of Brachyury, Xbra, and the zebrafish homologue, no tail (ntl), both function as transcription activators. The activation domains of both proteins map to their carboxy terminal regions, and we note that the activation domain is absent in two zebrafish Brachyury mutations, suggesting that it is required for gene function. A dominant-interfering Xbra construct was generated by replacing the activation domain of Xbra with the repressor domain of the Drosophila engrailed protein. Microinjection of RNA encoding this fusion protein allowed us to generate Xenopus and zebrafish embryos which show striking similarities to genetic mutants in mouse and fish. These results indicate that the function of Brachyury during vertebrate gastrulation is to activate transcription of mesoderm-specific genes. Additional experiments show that Xbra transcription activation is required for regulation of Xbra itself in dorsal, but not ventral, mesoderm. The approach described in this paper, in which the DNA-binding domain of a transcription activator is fused to the engrailed repressor domain, should assist in the analysis of other Xenopus and zebrafish transcription factors.

scholarly journals A high-throughput screen for transcription activation domains reveals their sequence characteristics and permits reliable prediction by deep learning

2019 ◽  
Author(s):  
Ariel Erijman ◽  
Lukasz Kozlowski ◽  
Salma Sohrabi-Jahromi ◽  
James Fishburn ◽  
Linda Warfield ◽  
...  
Keyword(s):  
Amino Acid ◽  
Transcription Activation ◽  
Amino Acid Sequences ◽  
Large Set ◽  
Hydrophobic Residues ◽  
Activation Domains ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Protein Interfaces ◽  
Wide Range

AbstractTranscription activation domains (ADs) are encoded by a wide range of seemingly unrelated amino acid sequences, making it difficult to recognize features that permit their dynamic behavior, fuzzy interactions and target specificity. We screened a large set of random 30-mer peptides for AD function and trained a deep neural network (ADpred) on the AD-positive and negative sequences. ADpred correctly identifies known ADs within protein sequences and accurately predicts the consequences of mutations. We show that functional ADs are (1) located within intrinsically disordered regions with biased amino acid composition, (2) contain clusters of hydrophobic residues near acidic side chains, (3) are enriched or depleted for particular dipeptide sequences, and (4) have higher helical propensity than surrounding regions. Taken together, our findings fit the model of “fuzzy” binding through hydrophobic protein-protein interfaces, where activator-coactivator binding takes place in a dynamic hydrophobic environment rather than through combinations of sequence-specific interactions.

scholarly journals Transcription activation domains of the yeast factors Met4 and Ino2: tandem activation domains with properties similar to the yeast Gcn4 activator

2017 ◽  
Author(s):  
Derek Pacheco ◽  
Linda warfield ◽  
Michelle Brajcich ◽  
Hannah Robbins ◽  
Jie Luo ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Transcription Activation ◽  
Binding Studies ◽  
Eukaryotic Transcription ◽  
Activation Domains ◽  
Conserved Sequence ◽  
Functional Studies ◽  
Intrinsically Disordered ◽  
Binding Domains ◽  
Optimal Function

AbstractEukaryotic transcription activation domains (ADs) are intrinsically disordered polypeptides that typically interact with coactivator complexes, leading to stimulation of transcription initiation, elongation and chromatin modifications. Here we examine the properties of two strong and conserved yeast ADs: Met4 and Ino2. Both factors have tandem ADs that were identified by conserved sequence and functional studies. While AD function from both factors depends on hydrophobic residues, Ino2 further requires key conserved acidic and polar residues for optimal function. Binding studies show that the ADs bind multiple Med15 activator binding domains (ABDs) with a similar order of micromolar affinity, and similar but distinct thermodynamic properties. Protein crosslinking shows that no unique complex is formed upon Met4-Med15 binding. Rather, we observed heterogeneous AD-ABD contacts with nearly every possible AD-ABD combination. Many of these properties are similar to those observed with the yeast activator Gcn4, which forms a large heterogeneous, dynamic, and fuzzy complex with Med15. We suggest that this molecular behavior is common among eukaryotic activators.

Dimerization through the helix-loop-helix motif enhances phosphorylation of the transcription activation domains of myogenin

1994 ◽  
Vol 14 (9) ◽  
pp. 6232-6243
Author(s):  
J Zhou ◽  
E N Olson
Keyword(s):  
Transcriptional Activity ◽  
Transcription Activation ◽  
Bhlh Protein ◽  
Specific Gene ◽  
Phosphorylation Sites ◽  
Gene Products ◽  
Activation Domains ◽  
Helix Loop Helix ◽  
Carboxyl Terminal ◽  
Bhlh Proteins

The muscle-specific basic helix-loop-helix (bHLH) protein myogenin activates muscle transcription by binding to target sequences in muscle-specific promoters and enhancers as a heterodimer with ubiquitous bHLH proteins, such as the E2A gene products E12 and E47. We show that dimerization with E2A products potentiates phosphorylation of myogenin at sites within its amino- and carboxyl-terminal transcription activation domains. Phosphorylation of myogenin at these sites was mediated by the bHLH region of E2A products and was dependent on dimerization but not on DNA binding. Mutations of the dimerization-dependent phosphorylation sites resulted in enhanced transcriptional activity of myogenin, suggesting that their phosphorylation diminishes myogenin's transcriptional activity. The ability of E2A products to potentiate myogenin phosphorylation suggests that dimerization induces a conformational change in myogenin that unmasks otherwise cryptic phosphorylation sites or that E2A proteins recruit a kinase for which myogenin is a substrate. That phosphorylation of these dimerization-dependent sites diminished myogenin's transcriptional activity suggests that these sites are targets for a kinase that interferes with muscle-specific gene expression.

scholarly journals A High-Throughput Screen for Transcription Activation Domains Reveals Their Sequence Features and Permits Prediction by Deep Learning

2020 ◽  
Vol 79 (6) ◽  
pp. 1066
Author(s):  
Ariel Erijman ◽  
Lukasz Kozlowski ◽  
Salma Sohrabi-Jahromi ◽  
James Fishburn ◽  
Linda Warfield ◽  
...  
Keyword(s):  
Deep Learning ◽  
High Throughput ◽  
Transcription Activation ◽  
High Throughput Screen ◽  
Activation Domains ◽  
Sequence Features

scholarly journals Fused protein domains inhibit DNA binding by LexA.

1992 ◽  
Vol 12 (7) ◽  
pp. 3006-3014 ◽  
Author(s):  
E A Golemis ◽  
R Brent
Keyword(s):  
Dna Binding ◽  
Protein Interactions ◽  
In Vitro Assays ◽  
Dimerization Domain ◽  
Activation Domains ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Operator Binding

Many studies of transcription activation employ fusions of activation domains to DNA binding domains derived from the bacterial repressor LexA and the yeast activator GAL4. Such studies often implicitly assume that DNA binding by the chimeric proteins is equivalent to that of the protein donating the DNA binding moiety. To directly investigate this issue, we compared operator binding by a series of LexA-derivative proteins to operator binding by native LexA, by using both in vivo and in vitro assays. We show that operator binding by many proteins such as LexA-Myc, LexA-Fos, and LexA-Bicoid is severely impaired, while binding of other LexA-derivative proteins, such as those that carry bacterially encoded acidic sequences ("acid blobs"), is not. Our results also show that DNA binding by LexA derivatives that contain the LexA carboxy-terminal dimerization domain (amino acids 88 to 202) is considerably stronger than binding by fusions that lack it and that heterologous dimerization motifs cannot substitute for the LexA88-202 function. These results suggest the need to reevaluate some previous studies of activation that employed LexA derivatives and modifications to recent experimental approaches that use LexA and GAL4 derivatives to detect and study protein-protein interactions.

scholarly journals Mapping the functional anatomy of Orai1 transmembrane domains for CRAC channel gating

2018 ◽  
Vol 115 (22) ◽  
pp. E5193-E5202 ◽  
Author(s):  
Priscilla S.-W. Yeung ◽  
Megumi Yamashita ◽  
Christopher E. Ing ◽  
Régis Pomès ◽  
Douglas M. Freymann ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Mutational Analysis ◽  
Channel Gating ◽  
Functional Anatomy ◽  
Transmembrane Domains ◽  
Hydrophobic Residues ◽  
Binding Domains ◽  
Allosteric Interactions ◽  
Crac Channel ◽  
Dynamics Simulations

Store-operated Orai1 channels are activated through a unique inside-out mechanism involving binding of the endoplasmic reticulum Ca2+ sensor STIM1 to cytoplasmic sites on Orai1. Although atomic-level details of Orai structure, including the pore and putative ligand binding domains, are resolved, how the gating signal is communicated to the pore and opens the gate is unknown. To address this issue, we used scanning mutagenesis to identify 15 residues in transmembrane domains (TMs) 1–4 whose perturbation activates Orai1 channels independently of STIM1. Cysteine accessibility analysis and molecular-dynamics simulations indicated that constitutive activation of the most robust variant, H134S, arises from a pore conformational change that opens a hydrophobic gate to augment pore hydration, similar to gating evoked by STIM1. Mutational analysis of this locus suggests that H134 acts as steric brake to stabilize the closed state of the channel. In addition, atomic packing analysis revealed distinct functional contacts between the TM1 pore helix and the surrounding TM2/3 helices, including one set mediated by a cluster of interdigitating hydrophobic residues and another by alternative ridges of polar and hydrophobic residues. Perturbing these contacts via mutagenesis destabilizes STIM1-mediated Orai1 channel gating, indicating that these bridges between TM1 and the surrounding TM2/3 ring are critical for conveying the gating signal to the pore. These findings help develop a framework for understanding the global conformational changes and allosteric interactions between topologically distinct domains that are essential for activation of Orai1 channels.

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