A Minimal Transcription Activation Domain Consisting of a Specific Array of Aspartic Acid and Leucine Residues

Abstract

Transcriptional activation by the herpesvirus protein VP16 (= Vmw65, aTlF) is mediated by its C-terminal acidic activation domain. Using GAL4 fusion proteins, we have previously shown that a construct containing two tandem copies of a short eleven amino acid fragment derived from the VP16 domain (DALDDFDLDML, residues 437-447) activates transcription in mammalian cells with an efficiency comparable to a GAL4 fusion with the full VP16 activation domain (residues 413-490). Here we have mutagenized this eleven amino acid core sequence and find that a mutant sequence with little inherent activity can cooperate with a wildtype sequence to yield almost full activity. Moreover, greater activity is observed when the wildtype sequence is positioned at the distal, rather than the proximal, end of the fusion protein, indicating that the distal position facilitates contacts to the transcription apparatus. We have also further reduced the eleven amino acid activating sequence to shorter sequence motifs. Two copies of eight and seven amino acids (DALDDFDL and DDFDLDL, respectively), or four copies of the sequence motif DDFDL are required to reach the activation potential of two eleven amino acid motifs. Four copies of the sequence DDLDL still activate transcription strongly (up to two-thirds of DDFDL), indicating that an aromatic residue is not an essential feature of this type of activation domain. However, repetitions of DDL or DL do not yield activity. Thus the minimal requirement for transcriptional activation is the presence of a sequence of some fifteen to twenty amino acids consisting of a specific array of aspartic acid and leucine residues. The motif DDLDL could be a prototypic activation module of the acidic/ hydrophobic class of activation domains.

Identification of Seven Hydrophobic Clusters in GCN4 Making Redundant Contributions to Transcriptional Activation

“…In addition, F-45, F-67, F-69, L-89, Y-110, W-120, and F-124 are flanked by acidic residues (Fig. 1), a feature characteristic of critical hydrophobic residues in the activation domains of several acidic activators (45,60). One interesting possibility is that the acidic residues in GCN4 increase the solubility of the hydrophobic clusters; alternatively, charge repulsion among the acidic residues may inhibit the formation of stable structures by the isolated activation domain, and these charges would be neutralized by interaction with basic residues on the surface of one or more transcription factors (61).…”

“…It appears that the subdomains in these proteins have overlapping functions and that efficient activation requires only a critical number of functionally redundant subdomains. This interpretation is supported by the fact that essentially wild-type levels of activation have been achieved in artificial activators containing reiterated copies of a single subdomain (60,61,64).…”

The Gcn4p Activation Domain Interacts Specifically In Vitro with RNA Polymerase II Holoenzyme, TFIID, and the Adap-Gcn5p Coactivator Complex

“…It has been proposed that the multiple activation domains in these proteins have redundant functions and that efficient activation requires only a critical number of domains to be present in the activator. This interpretation is supported by the fact that wildtype levels of activation can be achieved with artificial activators containing reiterated copies of a single domain (7,77,81). There is also evidence that a single activator can function in multiple ways to stimulate transcription.…”

A Small Ubiquitin-related Modifier-interacting Motif Functions as the Transcriptional Activation Domain of Krüppel-like Factor 4

“…Transcription factors also contain distinct domains that regulate the transcriptional machinery by interacting with both general and specific transcription factors and regulators (1)(2)(3)(4). The acidic activation domain, for example, represents the largest class of transcriptional activation domains and contains acidic and hydrophobic amino acid residues crucial for protein-protein interaction (1)(2)(3)(4).…”