Towards a minimal motif for artificial transcriptional activators

Background: Most transcriptional activators minimally comprise two function al modules, one for DNA binding and the other for activation. Several activ ators also bear an oligomerization region and bind DNA as dimers or higher order oligomers. In a previous study we substituted these domains of a prot ein activator with synthetic counterparts [Mapp ct al., Proc. Natl. Acad, S ci. USA 97 (2000) 3930-3935]. An artificial transcriptional activator, 4.2 kDa in size, comprised of a DNA binding hairpin polyamide tethered to a 20 residue activating peptide (AH) was shown to stimulate promoter specific tr anscription [Mapp et al., Proc. Natl. Acad, Sci. USA 97 (2000) 3930-3935]. The question arises as to the general nature and the versatility of this mi nimal activator motif and whether smaller ligands can be designed which mai ntain potent activation function. Results: Here we have replaced the 20 amino acid AH peptide with eight or 1 6 residues derived from the activation domain of the potent viral activator VP16. The 16 residue activation module coupled to the polyamide activated transcription over two-fold better than the analogous AH conjugate. Alterin g the site of attachment of the activation module on the polyamide allowed reduction of the intervening linker from 36 atoms to eight without signific ant diminution of the activation potential. In this study we also exchanged the polyamide to target a different sequence without compromising the acti vation function further demonstrating the generality of this design. Conclusions: The polyamide activator conjugates described here represent a class of DNA binding ligands which are tethered to a second functional moie ty, viz. an activation domain, that recruits elements of the endogenous tra nscriptional machinery. Our results define the minimal structural elements required to construct artificial, small molecule activators. If such activa tors are cell-permeable and can be targeted to designated sites in the geno me, this series of conjugates may then serve as a tool to study mechanistic aspects of transcriptional regulation and eventually to modulate gene expr ession relevant to human diseases. (C) 2001 Elsevier Science Ltd. All right s reserved.