scholarly journals Residue R113 Is Essential for PhoP Dimerization and Function: a Residue Buried in the Asymmetric PhoP Dimer Interface Determined in the PhoPN Three-Dimensional Crystal Structure

2003 ◽  
Vol 185 (1) ◽  
pp. 262-273 ◽  
Author(s):  
Yinghua Chen ◽  
Catherine Birck ◽  
Jean-Pierre Samama ◽  
F. Marion Hulett
Keyword(s):  
Crystal Structure ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Salt Bridge ◽  
Pho Regulon ◽  
Dimer Interface ◽  
And Function ◽  
Negative Phenotype

ABSTRACT Bacillus subtilis PhoP is a member of the OmpR/PhoB family of response regulators that is directly required for transcriptional activation or repression of Pho regulon genes in conditions under which Pi is growth limiting. Characterization of the PhoP protein has established that phosphorylation of the protein is not essential for PhoP dimerization or DNA binding but is essential for transcriptional regulation of Pho regulon genes. DNA footprinting studies of PhoP-regulated promoters showed that there was cooperative binding between PhoP dimers at PhoP-activated promoters and/or extensive PhoP oligomerization 3′ of PhoP-binding consensus repeats in PhoP-repressed promoters. The crystal structure of PhoPN described in the accompanying paper revealed that the dimer interface between two PhoP monomers involves nonidentical surfaces such that each monomer in a dimer retains a second surface that is available for further oligomerization. A salt bridge between R113 on one monomer and D60 on another monomer was judged to be of major importance in the protein-protein interaction. We describe the consequences of mutation of the PhoP R113 codon to a glutamate or alanine codon and mutation of the PhoP D60 codon to a lysine codon. In vivo expression of either PhoPR113E, PhoPR113A, or PhoPD60K resulted in a Pho-negative phenotype. In vitro analysis showed that PhoPR113E was phosphorylated by PhoR (the cognate histidine kinase) but was unable to dimerize. Monomeric PhoPR113E∼P was deficient in DNA binding, contributing to the PhoPR113E in vivo Pho-negative phenotype. While previous studies emphasized that phosphorylation was essential for PhoP function, data reported here indicate that phosphorylation is not sufficient as PhoP dimerization or oligomerization is also essential. Our data support the physiological relevance of the residues of the asymmetric dimer interface in PhoP dimerization and function.

scholarly journals Functional Domains of the TOL Plasmid Transcription Factor XylS

2000 ◽  
Vol 182 (4) ◽  
pp. 1118-1126 ◽  
Author(s):  
Niilo Kaldalu ◽  
Urve Toots ◽  
Victor de Lorenzo ◽  
Mart Ustav
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Dependent Manner ◽  
Tol Plasmid ◽  
Terminal Fragment ◽  
Central Regions ◽  
Basic Features ◽  
Regulatory Functions

ABSTRACT The alkylbenzoate degradation genes of Pseudomonas putida TOL plasmid are positively regulated by XylS, an AraC family protein, in a benzoate-dependent manner. In this study, we used deletion mutants and hybrid proteins to identify which parts of XylS are responsible for the DNA binding, transcriptional activation, and benzoate inducibility. We found that a 112-residue C-terminal fragment of XylS binds specifically to the Pm operator in vitro, protects this sequence from DNase I digestion identically to the wild-type (wt) protein, and activates the Pm promoter in vivo. When overexpressed, that C-terminal fragment could activate transcription as efficiently as wt XylS. All the truncations, which incorporated these 112 C-terminal residues, were able to activate transcription at least to some extent when overproduced. Intactness of the 210-residue N-terminal portion was found to be necessary for benzoate responsiveness of XylS. Deletions in the N-terminal and central regions seriously reduced the activity of XylS and caused the loss of effector control, whereas insertions into the putative interdomain region did not change the basic features of the XylS protein. Our results confirm that XylS consists of two parts which probably interact with each other. The C-terminal domain carries DNA-binding and transcriptional activation abilities, while the N-terminal region carries effector-binding and regulatory functions.

Reconstitution of transcriptional activation domains by reiteration of short peptide segments reveals the modular organization of a glutamine-rich activation domain

1994 ◽  
Vol 14 (9) ◽  
pp. 6056-6067
Author(s):  
M Tanaka ◽  
W Herr
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Activation Domain ◽  
Activation Domains ◽  
Transcriptional Activation Domain ◽  
Pou Domain

The POU domain activator Oct-2 contains an N-terminal glutamine-rich transcriptional activation domain. An 18-amino-acid segment (Q18III) from this region reconstituted a fully functional activation domain when tandemly reiterated and fused to either the Oct-2 or GAL4 DNA-binding domain. A minimal transcriptional activation domain likely requires three tandem Q18III segments, because one or two tandem Q18III segments displayed little activity, whereas three to five tandem segments were active and displayed increasing activity with increasing copy number. As with natural Oct-2 activation domains, in our assay a reiterated activation domain required a second homologous or heterologous activation domain to stimulate transcription effectively when fused to the Oct-2 POU domain. These results suggest that there are different levels of synergy within and among activation domains. Analysis of reiterated activation domains containing mutated Q18III segments revealed that leucines and glutamines, but not serines or threonines, are critical for activity in vivo. Curiously, several reiterated activation domains that were inactive in vivo were active in vitro, suggesting that there are significant functional differences in our in vivo and in vitro assays. Reiteration of a second 18-amino-acid segment from the Oct-2 glutamine-rich activation domain (Q18II) was also active, but its activity was DNA-binding domain specific, because it was active when fused to the GAL4 than to the Oct-2 DNA-binding domain. The ability of separate short peptide segments derived from a single transcriptional activation domain to activate transcription after tandem reiteration emphasizes the flexible and modular nature of a transcriptional activation domain.

scholarly journals Reading and Function of a Histone Code Involved in Targeting Corepressor Complexes for Repression

2005 ◽  
Vol 25 (1) ◽  
pp. 324-335 ◽  
Author(s):  
Ho-Geun Yoon ◽  
Youngsok Choi ◽  
Philip A. Cole ◽  
Jiemin Wong
Keyword(s):  
Transcriptional Activation ◽  
Transcriptional Repression ◽  
Hormone Receptors ◽  
Critical Role ◽  
Pericentric Heterochromatin ◽  
Histone Code ◽  
Stable Association ◽  
And Function

ABSTRACT A central question in histone code theory is how various codes are recognized and utilized in vivo. Here we show that TBL1 and TBLR1, two WD-40 repeat proteins in the corepressor SMRT/N-CoR complexes, are functionally redundant and essential for transcriptional repression by unliganded thyroid hormone receptors (TR) but not essential for transcriptional activation by liganded TR. TBL1 and TBLR1 bind preferentially to hypoacetylated histones H2B and H4 in vitro and have a critical role in targeting the corepressor complexes to chromatin in vivo. We show that targeting SMRT/N-CoR complexes to the deiodinase 1 gene (D1) requires at least two interactions, one between unliganded TR and SMRT/N-CoR and the other between TBL1/TBLR1 and hypoacetylated histones. Neither interaction alone is sufficient for the stable association of the corepressor complexes with the D1 promoter. Our data support a feed-forward working model in which deacetylation exerted by initial unstable recruitment of SMRT/N-CoR complexes via their interaction with unliganded TR generates a histone code that serves to stabilize their own recruitment. Similarly, we find that targeting of the Sin3 complex to pericentric heterochromatin may also follow this model. Our studies provide an in vivo example that a histone code is not read independently but is recognized in the context of other interactions.

scholarly journals Regulation of the DNA-binding and transcriptional activities of Xenopus laevis NFI-X by a novel C-terminal domain.

1995 ◽  
Vol 15 (10) ◽  
pp. 5552-5562 ◽  
Author(s):  
E Roulet ◽  
M T Armentero ◽  
G Krey ◽  
B Corthésy ◽  
C Dreyer ◽  
...  
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Xenopus Laevis ◽  
Transcriptional Activation ◽  
Binding Activity ◽  
New Members ◽  
Binding Domains ◽  
Dna Binding Activity

The nuclear factor I (NFI) family consists of sequence-specific DNA-binding proteins that activate both transcription and adenovirus DNA replication. We have characterized three new members of the NFI family that belong to the Xenopus laevis NFI-X subtype and differ in their C-termini. We show that these polypeptides can activate transcription in HeLa and Drosophila Schneider line 2 cells, using an activation domain that is subdivided into adjacent variable and subtype-specific domains each having independent activation properties in chimeric proteins. Together, these two domains constitute the full NFI-X transactivation potential. In addition, we find that the X. laevis NFI-X proteins are capable of activating adenovirus DNA replication through their conserved N-terminal DNA-binding domains. Surprisingly, their in vitro DNA-binding activities are specifically inhibited by a novel repressor domain contained within the C-terminal part, while the dimerization and replication functions per se are not affected. However, inhibition of DNA-binding activity in vitro is relieved within the cell, as transcriptional activation occurs irrespective of the presence of the repressor domain. Moreover, the region comprising the repressor domain participates in transactivation. Mechanisms that may allow the relief of DNA-binding inhibition in vivo and trigger transcriptional activation are discussed.

scholarly journals Yeast Coactivator MBF1 Mediates GCN4-Dependent Transcriptional Activation

1998 ◽  
Vol 18 (9) ◽  
pp. 4971-4976 ◽  
Author(s):  
Ken-ichi Takemaru ◽  
Satoshi Harashima ◽  
Hitoshi Ueda ◽  
Susumu Hirose
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Dna Binding ◽  
Nuclear Receptor ◽  
Transcriptional Activation ◽  
Crucial Role ◽  
Tata Binding Protein ◽  
Binding Region

ABSTRACT Transcriptional coactivators play a crucial role in gene expression by communicating between regulatory factors and the basal transcription machinery. The coactivator multiprotein bridging factor 1 (MBF1) was originally identified as a bridging molecule that connects theDrosophila nuclear receptor FTZ-F1 and TATA-binding protein (TBP). The MBF1 sequence is highly conserved across species fromSaccharomyces cerevisiae to human. Here we provide evidence acquired in vitro and in vivo that yeast MBF1 mediates GCN4-dependent transcriptional activation by bridging the DNA-binding region of GCN4 and TBP. These findings indicate that the coactivator MBF1 functions by recruiting TBP to promoters where DNA-binding regulators are bound.

scholarly journals Synergistic transcriptional activation by CTF/NF-I and the estrogen receptor involves stabilized interactions with a limiting target factor.

1991 ◽  
Vol 11 (6) ◽  
pp. 2937-2945 ◽  
Author(s):  
E Martinez ◽  
Y Dusserre ◽  
W Wahli ◽  
N Mermod
Keyword(s):  
Estrogen Receptor ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Transcription Initiation ◽  
Limiting Factor ◽  
Gene Promoters ◽  
Protein Coding ◽  
Transcriptional Activation Domain

Transcription initiation at eukaryotic protein-coding gene promoters is regulated by a complex interplay of site-specific DNA-binding proteins acting synergistically or antagonistically. Here, we have analyzed the mechanisms of synergistic transcriptional activation between members of the CCAAT-binding transcription factor/nuclear factor I (CTF/NF-I) family and the estrogen receptor. By using cotransfection experiments with HeLa cells, we show that the proline-rich transcriptional activation domain of CTF-1, when fused to the GAL4 DNA-binding domain, synergizes with each of the two estrogen receptor-activating regions. Cooperative DNA binding between the GAL4-CTF-1 fusion and the estrogen receptor does not occur in vitro, and in vivo competition experiments demonstrate that both activators can be specifically inhibited by the overexpression of a proline-rich competitor, indicating that a common limiting factor is mediating their transcriptional activation functions. Furthermore, the two activators functioning synergistically are much more resistant to competition than either factor alone, suggesting that synergism between CTF-1 and the estrogen receptor is the result of a stronger tethering of the limiting target factor(s) to the two promoter-bound activators.

scholarly journals The structure of a class 3 nonsymbiotic plant haemoglobin fromArabidopsis thalianareveals a novel N-terminal helical extension

2014 ◽  
Vol 70 (5) ◽  
pp. 1411-1418 ◽  
Author(s):  
Brandon J. Reeder ◽  
Michael A. Hough
Keyword(s):  
Crystal Structure ◽  
Protein Sequence ◽  
Disulfide Bonds ◽  
External Environment ◽  
Tryptophan Residue ◽  
Central Cavity ◽  
Dimer Interface ◽  
Iron Coordination

Plant nonsymbiotic haemoglobins fall into three classes, each with distinct properties but all with largely unresolved physiological functions. Here, the first crystal structure of a class 3 nonsymbiotic plant haemoglobin, that fromArabidopsis thaliana, is reported to 1.77 Å resolution. The protein forms a homodimer, with each monomer containing a two-over-two α-helical domain similar to that observed in bacterial truncated haemoglobins. A novel N-terminal extension comprising two α-helices plays a major role in the dimer interface, which occupies the periphery of the dimer–dimer face, surrounding an open central cavity. The haem pocket contains a proximal histidine ligand and an open sixth iron-coordination site with potential for a ligand, in this structure hydroxide, to form hydrogen bonds to a tyrosine or a tryptophan residue. The haem pocket appears to be unusually open to the external environment, with another cavity spanning the entrance of the two haem pockets. The final 23 residues of the C-terminal domain are disordered in the structure; however, these domains in the functional dimer are adjacent and include the only two cysteine residues in the protein sequence. It is likely that these residues form disulfide bondsin vitroand it is conceivable that this C-terminal region may act in a putative complex with a partner moleculein vivo.

scholarly journals Simple biochemical features underlie transcriptional activation domain diversity and dynamic, fuzzy binding to Mediator

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Adrian L Sanborn ◽  
Benjamin T Yeh ◽  
Jordan T Feigerle ◽  
Cynthia V Hao ◽  
Raphael J L Townshend ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Structural Constraints ◽  
Sequence Motifs ◽  
Activation Domains ◽  
Activator Proteins ◽  
Transcriptional Activation Domain ◽  
Shape Complementarity ◽  
And Function

Gene activator proteins comprise distinct DNA-binding and transcriptional activation domains (ADs). Because few ADs have been described, we tested domains tiling all yeast transcription factors for activation in vivo and identified 150 ADs. By mRNA display, we showed that 73% of ADs bound the Med15 subunit of Mediator, and that binding strength was correlated with activation. AD-Mediator interaction in vitro was unaffected by a large excess of free activator protein, pointing to a dynamic mechanism of interaction. Structural modeling showed that ADs interact with Med15 without shape complementarity ('fuzzy' binding). ADs shared no sequence motifs, but mutagenesis revealed biochemical and structural constraints. Finally, a neural network trained on AD sequences accurately predicted ADs in human proteins and in other yeast proteins, including chromosomal proteins and chromatin remodeling complexes. These findings solve the longstanding enigma of AD structure and function and provide a rationale for their role in biology.

scholarly journals Scanning Mutagenesis of Mcm1: Residues Required for DNA Binding, DNA Bending, and Transcriptional Activation by a MADS-Box Protein

2000 ◽  
Vol 20 (1) ◽  
pp. 1-11 ◽  
Author(s):  
Thomas B. Acton ◽  
Janet Mead ◽  
Andrew M. Steiner ◽  
Andrew K. Vershon
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Serum Response Factor ◽  
Essential Gene ◽  
Dna Bending ◽  
Mads Box ◽  
Dna Interactions ◽  
Protein Dna Interactions

ABSTRACT MCM1 is an essential gene in the yeastSaccharomyces cerevisiae and is a member of the MADS-box family of transcriptional regulatory factors. To understand the nature of the protein-DNA interactions of this class of proteins, we have made a series of alanine substitutions in the DNA-binding domain of Mcm1 and examined the effects of these mutations in vivo and in vitro. Our results indicate which residues of Mcm1 are important for viability, transcriptional activation, and DNA binding and bending. Substitution of residues in Mcm1 which are highly conserved among the MADS-box proteins are lethal to the cell and abolish DNA binding in vitro. These positions have almost identical interactions with DNA in both the serum response factor-DNA and α2-Mcm1-DNA crystal structures, suggesting that these residues make up a conserved core of protein-DNA interactions responsible for docking MADS-box proteins to DNA. Substitution of residues which are not as well conserved among members of the MADS-box family play important roles in contributing to the specificity of DNA binding. These results suggest a general model of how MADS-box proteins recognize and bind DNA. We also provide evidence that the N-terminal extension of Mcm1 may have considerable conformational freedom, possibly to allow binding to different DNA sites. Finally, we have identified two mutants at positions which are critical for Mcm1-mediated DNA bending that have a slow-growth phenotype. This finding is consistent with our earlier results, indicating that DNA bending may have a role in Mcm1 function in the cell.

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