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.

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.

scholarly journals Evolution of the activation domain in a Hox transcription factor

2018 ◽  
Vol 62 (11-12) ◽  
pp. 745-753 ◽  
Author(s):  
Ying Liu ◽  
Annie Huang ◽  
Rebecca M. Booth ◽  
Gabriela Geraldo Mendes ◽  
Zabeena Merchant ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Intrinsically Disordered Protein ◽  
Transcription Activation ◽  
Specific Protein ◽  
Amino Acid Sequences ◽  
Activation Domain ◽  
Mutant Proteins ◽  
Activation Domains ◽  
Intrinsically Disordered

Linking changes in amino acid sequences to the evolution of transcription regulatory domains is often complicated by the low sequence complexity and high mutation rates of intrinsically disordered protein regions. For the Hox transcription factor Ultrabithorax (Ubx), conserved motifs distributed throughout the protein sequence enable direct comparison of specific protein regions, despite variations in the length and composition of the intervening sequences. In cell culture, the strength of transcription activation by Drosophila melanogaster Ubx correlates with the presence of a predicted helix within its activation domain. Curiously, this helix is not preserved in species more divergent than flies, suggesting the nature of transcription activation may have evolved. To determine whether this helix contributes to Drosophila Ubx function in vivo, wild-type and mutant proteins were ectopically expressed in the developing wing and the phenotypes evaluated. Helix mutations alter Drosophila Ubx activity in the developing wing, demonstrating its functional importance in vivo. The locations of activation domains in Ubx orthologues were identified by testing the ability of truncation mutants to activate transcription in yeast one-hybrid assays. In Ubx orthologues representing 540 million years of evolution, the ability to activate transcription varies substantially. The sequence and the location of the activation domains also differ. Consequently, analogous regions of Ubx orthologues change function over time, and may activate transcription in one species, but have no activity, or even inhibit transcription activation in another species. Unlike homeodomain-DNA binding, the nature of transcription activation by Ubx has substantially evolved.

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 Fungal Mediator Tail Subunits Contain Classical Transcriptional Activation Domains

2015 ◽  
Vol 35 (8) ◽  
pp. 1363-1375 ◽  
Author(s):  
Zhongle Liu ◽  
Lawrence C. Myers
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Weak Interactions ◽  
Primary Role ◽  
Eukaryotic Transcription ◽  
Content Type ◽  
Activation Domains ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Transcriptional Activation Domains

Classical activation domains within DNA-bound eukaryotic transcription factors make weak interactions with coactivator complexes, such as Mediator, to stimulate transcription. How these interactions stimulate transcription, however, is unknown. The activation of reporter genes by artificial fusion of Mediator subunits to DNA binding domains that bind to their promoters has been cited as evidence that the primary role of activators is simply to recruit Mediator. We have identified potent classical transcriptional activation domains in the C termini of several tail module subunits ofSaccharomyces cerevisiae,Candida albicans, andCandida dubliniensisMediator, while their N-terminal domains are necessary and sufficient for their incorporation into Mediator but do not possess the ability to activate transcription when fused to a DNA binding domain. This suggests that Mediator fusion proteins actually are functioning in a manner similar to that of a classical DNA-bound activator rather than just recruiting Mediator. Our finding that deletion of the activation domains ofS. cerevisiaeMed2 and Med3, as well asC. dubliniensisTlo1 (a Med2 ortholog), impairs the induction of certain genes shows these domains function at native promoters. Activation domains within coactivators are likely an important feature of these complexes and one that may have been uniquely leveraged by a common fungal pathogen.

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.

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 Structural insights into the interaction of helicase and primase in Mycobacterium tuberculosis

2018 ◽  
Vol 475 (21) ◽  
pp. 3493-3509 ◽  
Author(s):  
Dhakaram Pangeni Sharma ◽  
Ramachandran Vijayan ◽  
Syed Arif Abdul Rehman ◽  
Samudrala Gourinath
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Replication Initiation ◽  
Binding Studies ◽  
Functional Conservation ◽  
Terminal Domain ◽  
Binding Domains ◽  
Initiation System ◽  
Helical Hairpin ◽  
Helicase Dnab ◽  
Species Specific

The helicase–primase interaction is an essential event in DNA replication and is mediated by the highly variable C-terminal domain of primase (DnaG) and N-terminal domain of helicase (DnaB). To understand the functional conservation despite the low sequence homology of the DnaB-binding domains of DnaGs of eubacteria, we determined the crystal structure of the helicase-binding domain of DnaG from Mycobacterium tuberculosis (MtDnaG-CTD) and did so to a resolution of 1.58 Å. We observed the overall structure of MtDnaG-CTD to consist of two subdomains, the N-terminal globular region (GR) and the C-terminal helical hairpin region (HHR), connected by a small loop. Despite differences in some of its helices, the globular region was found to have broadly similar arrangements across the species, whereas the helical hairpins showed different orientations. To gain insights into the crucial helicase–primase interaction in M. tuberculosis, a complex was modeled using the MtDnaG-CTD and MtDnaB-NTD crystal structures. Two nonconserved hydrophobic residues (Ile605 and Phe615) of MtDnaG were identified as potential key residues interacting with MtDnaB. Biosensor-binding studies showed a significant decrease in the binding affinity of MtDnaB-NTD with the Ile605Ala mutant of MtDnaG-CTD compared with native MtDnaG-CTD. The loop, connecting the two helices of the HHR, was concluded to be largely responsible for the stability of the DnaB–DnaG complex. Also, MtDnaB-NTD showed micromolar affinity with DnaG-CTDs from Escherichia coli and Helicobacter pylori and unstable binding with DnaG-CTD from Vibrio cholerae. The interacting domains of both DnaG and DnaB demonstrate the species-specific evolution of the replication initiation system.

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