Structural and functional properties of the N transcriptional activation domain of thyroid transcription factor-1: similarities with the acidic activation domains

Abstract

The thyroid transcription factor 1 (TTF-1) is a tissue-specific transcription factor involved in the development of thyroid and lung. TTF-1 contains two transcriptional activation domains (N and C domain). The primary amino acid sequence of the N domain does not show any typical characteristic of known transcriptional activation domains. In aqueous solution the N domain exists in a random-coil conformation. The increase of the milieu hydrophobicity, by the addition of trifluoroethanol, induces a considerable gain of alpha-helical structure. Acidic transcriptional activation domains are largely unstructured in solution, but, under hydrophobic conditions, folding into alpha-helices or beta-strands can be induced. Therefore our data indicate that the inducibility of alpha-helix by hydrophobic conditions is a property not restricted to acidic domains. Co-transfections experiments indicate that the acidic domain of herpes simplex virus protein VP16 (VP16) and the TTF-1 N domain are interchangeable and that a chimaeric protein, which combines VP16 linked to the DNA-binding domain of TTF-1, undergoes the same regulatory constraints that operate for the wild-type TTF-1. In addition, we demonstrate that the TTF-1 N domain possesses two typical properties of acidic activation domains: TBP (TATA-binding protein) binding and ability to activate transcription in yeast. Accordingly, the TTF-1 N domain is able to squelch the activity of the p65 acidic domain. Altogether, these structural and functional data suggest that a non-acidic transcriptional activation domain (TTF-1 N domain) activates transcription by using molecular mechanisms similar to those used by acidic domains. TTF-1 N domain and acidic domains define a family of proteins whose common property is to activate transcription through the use of mechanisms largely conserved during evolutionary development.

Local Structural Elements in the Mostly Unstructured Transcriptional Activation Domain of Human p53

“…One possible structural change is conversion of the two nascent turns found in the unbound state into an amphiphatic helix due to structural restraints imposed by a target protein. The fact that overall helical content of several AADs increases upon addition of hydrophobic solvents (9,19,20,38,39) 37 linker between the two helices. Such a speculation is supported by the fact that mutation of two prolines, which are neither acidic nor conserved hydrophobic residues, in the proposed linker to hydrophilic residues results in significant (ϳ25%) activity loss (9).…”

“…The observation that these proteins may function without necessarily relying on tertiary structure has in fact suggested establishment of a new view on protein structure (30,37). The unbound full-length p53 TAD, largely unstructured in aqueous solution, contains a small fraction of secondary structure (9), as do many other TADs (28,38,39). In order to fully understand the potential ramification of such minimal secondary structures for transcriptional activity, we have carried out a detailed multidimensional NMR study on a uniformly 15 N-labeled full-length p53 TAD.…”

Transcriptional Repressor DREAM Interacts with Thyroid Transcription Factor-1 and Regulates Thyroglobulin Gene Expression

“…For pull-down studies, TTF-1 and its truncated forms were 35 Slabeled in vitro using the transcription/translation T7-TNT system (Promega). Equimolar amounts of recombinant GST and GST-DREAM proteins (ϳ15-20 pmol) bound to glutathione-Sepharose (Amersham Biosciences) were incubated with the labeled proteins in interaction buffer (20 mM potassium Hepes, pH 7.5, 10% glycerol, 150 mM KCl, 2 mM MgCl 2 , 0.5 mM EGTA, 1 mM dithiothreitol, 0.1% Nonidet P-40, 0.5% Blotto (Bio-Rad), and protease inhibitor mixture (Calbiochem)).…”

“…4B). To investigate the domains in TTF-1 responsible for the interaction with DREAM we used pull-down assays using a GST-DREAM fusion protein and 35 S-labeled in vitro-translated TTF-1 full-length protein or truncated forms (Fig. 5A).…”

Characterization of the Interactions between the Nucleoprotein and the Phosphoprotein of Henipavirus

“…7, A and B). The solvent TFE is widely used as an empirical probe of hidden structural propensities of peptides and proteins, as it mimics the hydrophobic environment experienced by proteins in protein-protein interactions (87)(88)(89). Both proteins show an increasing gain of ␣-helicity upon the addition of TFE, as indicated by the characteristic maximum at 190 nm and double minima at 208 and 222 nm (Fig.…”