scholarly journals The glucocorticoid receptor DNA-binding domain recognizes RNA hairpin structures with high affinity

2019 ◽  
Vol 47 (15) ◽  
pp. 8180-8192 ◽  
Author(s):  
Nicholas V Parsonnet ◽  
Nickolaus C Lammer ◽  
Zachariah E Holmes ◽  
Robert T Batey ◽  
Deborah S Wuttke
Keyword(s):  
Dna Binding ◽  
Glucocorticoid Receptor ◽  
Noncoding Rna ◽  
Rna Binding ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Diverse Range ◽  
High Affinity ◽  
Rna Hairpin

AbstractThe glucocorticoid receptor (GR) binds the noncoding RNA Gas5 via its DNA-binding domain (DBD) with functional implications in pro-apoptosis signaling. Here, we report a comprehensive in vitro binding study where we have determined that GR-DBD is a robust structure-specific RNA-binding domain. GR-DBD binds to a diverse range of RNA hairpin motifs, both synthetic and biologically derived, with apparent mid-nanomolar affinity while discriminating against uniform dsRNA. As opposed to dimeric recognition of dsDNA, GR-DBD binds to RNA as a monomer and confers high affinity primarily through electrostatic contacts. GR-DBD adopts a discrete RNA-bound state, as assessed by NMR, distinct from both free and DNA-bound. NMR and alanine mutagenesis suggest a heightened involvement of the C-terminal α-helix of the GR-DBD in RNA-binding. RNA competes for binding with dsDNA and occurs in a similar affinity range as dimer binding to the canonical DNA element. Given the prevalence of RNA hairpins within the transcriptome, our findings strongly suggest that many RNAs have potential to impact GR biology.

scholarly journals Molecular Determinants of Glucocorticoid Receptor Mobility in Living Cells: the Importance of Ligand Affinity

2003 ◽  
Vol 23 (6) ◽  
pp. 1922-1934 ◽  
Author(s):  
Marcel J. M. Schaaf ◽  
John A. Cidlowski
Keyword(s):  
Dna Binding ◽  
Glucocorticoid Receptor ◽  
Ligand Binding ◽  
Conformational Change ◽  
Binding Domain ◽  
Ligand Binding Domain ◽  
Dependent Manner ◽  
Dna Binding Domain ◽  
Ligand Affinity ◽  
High Affinity

ABSTRACT The actions of glucocorticoids are mediated by the glucocorticoid receptor (GR), which is activated upon ligand binding, and can alter the expression of target genes either by transrepression or transactivation. We have applied FRAP (fluorescence recovery after photobleaching) to quantitatively assess the mobility of the yellow fluorescent protein (YFP)-tagged human GR α-isoform (hGRα) in the nucleus of transiently transfected COS-1 cells and to elucidate determinants of its mobility. Addition of the high-affinity agonist dexamethasone markedly decreases the mobility of the receptor in a concentration-dependent manner, whereas low-affinity ligands like corticosterone decrease the mobility to a much lesser extent. Analysis of other hGRα ligands differing in affinity suggests that it is the affinity of the ligand that is a major determinant of the decrease in mobility. Similar results were observed for two hGRα antagonists, the low-affinity antagonist ZK98299 and the high-affinity antagonist RU486. The effect of ligand affinity on mobility was confirmed with the hGRα mutant Q642V, which has an altered affinity for triamcinolone acetonide, dexamethasone, and corticosterone. Analysis of hGRα deletion mutants indicates that both the DNA-binding domain and the ligand-binding domain of the receptor are required for a maximal ligand-induced decrease in receptor mobility. Interestingly, the mobility of transfected hGRα differs among cell types. Finally, the proteasome inhibitor MG132 immobilizes a subpopulation of unliganded receptors, via a mechanism requiring the DNA-binding domain and the N-terminal part of the ligand-binding domain. Ligand binding makes the GR resistant to the immobilizing effect of MG132, and this effect depends on the affinity of the ligand. Our data suggest that ligand binding induces a conformational change of the receptor which is dependent on the affinity of the ligand. This altered conformation decreases the mobility of the receptor, probably by targeting the receptor to relatively immobile nuclear domains with which it transiently associates. In addition, this conformational change blocks immobilization of the receptor by MG132.

scholarly journals In vitro translation of androgen receptor cRNA results in an activated androgen receptor protein

1993 ◽  
Vol 296 (1) ◽  
pp. 161-167 ◽  
Author(s):  
G G J M Kuiper ◽  
P E de Ruiter ◽  
J Trapman ◽  
G Jenster ◽  
A O Brinkmann
Keyword(s):  
Androgen Receptor ◽  
Dna Binding ◽  
Binding Domain ◽  
In Vitro Translation ◽  
Receptor Protein ◽  
Dna Binding Domain ◽  
In Vitro Synthesis ◽  
High Affinity ◽  
Responsive Element

Translation of androgen receptor (AR) cRNA in a reticulocyte lysate and subsequent analysis of the translation products by SDS/PAGE showed a protein with an apparent molecular mass of 108 kDa. Scatchard-plot analysis revealed a single binding component with high affinity for R1881 (Kd = 0.3 nM). All AR molecules synthesized specifically bound steroid. No evidence for AR phosphorylation during in vitro synthesis was found. When AR was labelled with [3H]R1881 and analysed on sucrose-density gradients, a complex of approx. 6 S was observed. The complex was shifted to a higher sedimentation coefficient after incubation with a monoclonal AR antibody directed against an epitope in the DNA-binding domain. In the presence as well as the absence of hormone, AR molecules were able to bind to DNA-cellulose without an activation step. Gel retardation assays revealed that the AR forms complexes with a DNA element containing glucocorticoid-responsive element/androgen-responsive element sequences. Receptor-DNA interactions were stabilized by different polyclonal antibodies directed against either the N- or C-terminal part of the AR and were abolished by an antibody directed against the DNA-binding domain of the receptor. In conclusion, translation of AR cRNA in vitro yields an activated AR protein which binds steroid with high affinity. It is proposed that AR antibodies enhance AR-DNA binding by stabilizing AR dimers when bound to DNA.

scholarly journals Fos is a preferential target of glucocorticoid receptor inhibition of AP-1 activity in vitro.

1993 ◽  
Vol 13 (6) ◽  
pp. 3782-3791 ◽  
Author(s):  
T K Kerppola ◽  
D Luk ◽  
T Curran
Keyword(s):  
Dna Binding ◽  
Glucocorticoid Receptor ◽  
Leucine Zipper ◽  
Molecular Mechanisms ◽  
Hormone Receptors ◽  
Transcription Activation ◽  
Nuclear Hormone Receptor ◽  
Binding Domain ◽  
Dna Binding Domain

Several regulatory interactions between the AP-1 and the nuclear hormone receptor families of transcription factors have been reported. However, the molecular mechanisms that underlie these interactions remain unknown, and models derived from transient-transfection experiments are contradictory. We have investigated the effect of the purified glucocorticoid receptor (GR) DNA-binding domain (GR residues 440 to 533 [GR440-533]) on DNA binding and transcription activation by Fos-Jun heterodimers and Jun homodimers. GR440-533 differentially inhibited DNA binding and transcription activation by Fos-Jun heterodimers. Inhibition of Jun homodimers required a 10-fold-higher concentration of GR440-533. An excess of Fos monomers protected Fos-Jun heterodimers from inhibition by GR440-533. Surprisingly, regions outside the leucine zipper and basic region were required for GR inhibition of Fos and Jun DNA binding. The region of GR440-533 required for inhibition of Fos-Jun DNA binding was localized to the zinc finger DNA-binding domain. However, inhibition of Fos-Jun DNA binding was independent of DNA binding by GR440-533. GR440-533 also differentially inhibited Fos-Jun heterodimer binding to the proliferin plfG element. Differential inhibition of DNA binding by different AP-1 family complexes provides a potential mechanism for the diverse interactions between nuclear hormone receptors and AP-1 family proteins at different promoters and in different cell types.

Fos is a preferential target of glucocorticoid receptor inhibition of AP-1 activity in vitro

1993 ◽  
Vol 13 (6) ◽  
pp. 3782-3791 ◽  
Author(s):  
T K Kerppola ◽  
D Luk ◽  
T Curran
Keyword(s):  
Dna Binding ◽  
Glucocorticoid Receptor ◽  
Leucine Zipper ◽  
Molecular Mechanisms ◽  
Hormone Receptors ◽  
Transcription Activation ◽  
Nuclear Hormone Receptor ◽  
Binding Domain ◽  
Dna Binding Domain

Several regulatory interactions between the AP-1 and the nuclear hormone receptor families of transcription factors have been reported. However, the molecular mechanisms that underlie these interactions remain unknown, and models derived from transient-transfection experiments are contradictory. We have investigated the effect of the purified glucocorticoid receptor (GR) DNA-binding domain (GR residues 440 to 533 [GR440-533]) on DNA binding and transcription activation by Fos-Jun heterodimers and Jun homodimers. GR440-533 differentially inhibited DNA binding and transcription activation by Fos-Jun heterodimers. Inhibition of Jun homodimers required a 10-fold-higher concentration of GR440-533. An excess of Fos monomers protected Fos-Jun heterodimers from inhibition by GR440-533. Surprisingly, regions outside the leucine zipper and basic region were required for GR inhibition of Fos and Jun DNA binding. The region of GR440-533 required for inhibition of Fos-Jun DNA binding was localized to the zinc finger DNA-binding domain. However, inhibition of Fos-Jun DNA binding was independent of DNA binding by GR440-533. GR440-533 also differentially inhibited Fos-Jun heterodimer binding to the proliferin plfG element. Differential inhibition of DNA binding by different AP-1 family complexes provides a potential mechanism for the diverse interactions between nuclear hormone receptors and AP-1 family proteins at different promoters and in different cell types.

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 Cell Cycle Localization, Dimerization, and Binding Domain Architecture of the Telomere Protein cPot1

2004 ◽  
Vol 24 (5) ◽  
pp. 2091-2102 ◽  
Author(s):  
Chao Wei ◽  
Carolyn M. Price
Keyword(s):  
Cell Cycle ◽  
Dna Binding ◽  
Binding Site ◽  
Domain Architecture ◽  
Binding Domain ◽  
Binding Motif ◽  
Dna Binding Domain ◽  
Telomere Protein ◽  
Ob Fold

ABSTRACT Pot1 is a single-stranded-DNA-binding protein that recognizes telomeric G-strand DNA. It is essential for telomere capping in Saccharomyces pombe and regulates telomere length in humans. Human Pot1 also interacts with proteins that bind the duplex region of the telomeric tract. Thus, like Cdc13 from S. cerevisiae, Pot 1 may have multiple roles at the telomere. We show here that endogenous chicken Pot1 (cPot1) is present at telomeres during periods of the cell cycle when t loops are thought to be present. Since cPot1 can bind internal loops and directly adjacent DNA-binding sites, it is likely to fully coat and protect both G-strand overhangs and the displaced G strand of a t loop. The minimum binding site of cPot1 is double that of the S. pombe DNA-binding domain. Although cPot can self associate, dimerization is not required for DNA binding and hence does not explain the binding-site duplication. Instead, the DNA-binding domain appears to be extended to contain a second binding motif in addition to the conserved oligonucleotide-oligosaccharide (OB) fold present in other G-strand-binding proteins. This second motif could be another OB fold. Although dimerization is inefficient in vitro, it may be regulated in vivo and could promote association with other telomere proteins and/or telomere compaction.

scholarly journals Structure of the XPA DNA binding domain and RPA high-affinity DNA binding domains on a model NER substrate

2019 ◽  
Vol 75 (a1) ◽  
pp. a203-a203
Author(s):  
Walter J. Chazin ◽  
Agnieszka M. Topolska-Woś ◽  
Norie Sugitani ◽  
John J. Cordoba ◽  
Hyun Suk Kim ◽  
...  
Keyword(s):  
Dna Binding ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Dna Binding Domains ◽  
High Affinity ◽  
Binding Domains
Sign in / Sign up

Export Citation Format

Share Document