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Transcriptional activation domains share little sequence homology and generally lack folded structures in the absence of their targets, aspects that have rendered activation domains difficult to characterize. Here, a combination of biochemical and nuclear magnetic resonance experiments demonstrates that the activation domain of the tumor suppressor p53 has an FXXΦΦ motif (F, Phe; X, any amino acids; Φ, hydrophobic residues) that folds into an α-helix upon binding to one of its targets, hTAF II 31 (a human TFIID TATA box-binding protein-associated factor). MDM2, the cellular attenuator of p53, discriminates the FXXΦΦ motif of p53 from those of NF-κB p65 and VP16 and specifically inhibits p53 activity. Our studies support the notion that the FXXΦΦ sequence is a general α-helical recognition motif for hTAF II 31 and provide insights into the mechanistic basis for regulation of p53 function.

In eukaryotes, sequence-specific DNA-binding proteins activate gene expression by recruiting the transcriptional apparatus and chromatin remodeling proteins to the promoter through protein-protein contacts. In many instances, the connection between DNA-binding proteins and the transcriptional apparatus is established through the intermediacy of adapter proteins known as coactivators. Here we describe synthetic molecules with low molecular weight that act as transcriptional coactivators. We demonstrate that a completely nonnatural activation domain in one such molecule is capable of stimulating transcription in vitro and in vivo . The present strategy provides a means of gaining external control over gene activation through intervention using small molecules.

External control of gene expression by small synthetic molecules represents a challenge in chemistry. Here we discuss the synthetic aspects of small molecule transcription factor mimics including a molecule we call STF1 (1). STF1 stimulates the transcription of a reporter gene controlled by specific DNA elements and behaves just as a naturally occurring transcription factor. However, STF1 had limited cell permeability, even though the hairpin-polyamide-FITC conjugate and the wrenchnolol (2) are cell permeable as separate compounds. Systematic synthetic exploration of STF1 analogs (STFs), by optimizing the physical properties of the molecule, is expected to increase its cell permeability for biological studies.

Activation domains are functional modules that enable sequence-specific DNA binding proteins to stimulate transcription. The structural basis for the function of activation domains is poorly understood. A combination of nuclear magnetic resonance (NMR) and biochemical experiments revealed that the minimal acidic activation domain of the herpes simplex virus VP16 protein undergoes an induced transition from random coil to α helix upon binding to its target protein, hTAF II 31 (a human TFIID TATA box – binding protein-associated factor). Identification of the two hydrophobic residues that make nonpolar contacts suggests a general recognition motif of acidic activation domains for hTAF II 31.

This study demonstrates unique reactivity of the C-1027 chromophore toward a tRNAphe. In the presence of Mg2+ ions where the tRNAphe attains a stable three-dimensional structure, the enediyne chromophore exhibits high cleavage selectivity to the anticodon arm. The present reactivity of the C-1027 chromophore is useful in development of new chemical probes for mapping of tertiary RNA structure. Considering the paucity of RNA repair mechanisms, RNA may be also an important biological target for certain enediyne antibiotics.

We present evidence that the enediyne antitumor antibiotic dynemicin recognizes and cleaves conformationally flexible regions of DNA. This is based on specific strand breaks at mismatches, bulges, and nicks as determined by high-resolution sequencing gels. On the basis of the weak association constant of dynemicin for DNA, it is expected that these more flexible regions would be preferred sites. In addition, the DNA unwinding behavior of dynemicin and the absorption spectrum of the dynemicin-DNA complex are strongly indicative of its intercalative binding with DNA. The results allow us to propose dynemicin as a molecular wedge that binds to the DNA by intercalating into the minor-groove side of conformationally flexible regions of DNA. Presumably, DNA local flexibility is able to create an open pocket in the minor groove, permits facile intercalation of dynemicin, and then increases the chances of its DNA damaging event. Implications for the biological action of dynemicin have also been discussed.

Summary Elsamicin A is an antitumor antibiotic with fascinating chemical structure and a good candidate for pharmaceutical development. Molecular mechanism of DNA backbone cleavage mediated by Fe(II)-elsamicin A has been examined. Product analysis using DNA sequencing gels and HPLC reveals the production of damaged DNA fragments bearing 3′-/5′-phosphate and 3′-phosphoglycolate termini associated with formation of free base. In addition, hydrazine-trapping experiments indicate that C-4′ hydroxylated abasic sites are formed concomitant with DNA degradation by Fe(II)-elsamicin A. The results lead to the conclusion that the hydroxyl radical formed in Fe(II)-elsamicin A plus dithiothreitol system oxidizes the deoxyribose moiety via hydrogen abstraction predominantly at the C-4′ carbon of the deoxyribose backbone and ultimately produces strand breakage of DNA.

A large collection of bioactive compounds with diverse biological effects can be used as probes to elucidate new biological mechanisms that influence a particular cellular process. Here we analyze the effects of 880 well-known small-molecule bioactives or drugs on the insulin-induced adipogenesis of 3T3-L1 fibroblasts, a cell-culture model of fat cell differentiation. Our screen identified 86 compounds as modulators of the adipogenic differentiation of 3T3-L1 cells. Examination of their chemical and pharmacological information revealed that antihistamine drugs with distinct chemical scaffolds inhibit differentiation. Histamine H1 receptor is expressed in 3T3-L1 cells, and its knockdown by small interfering RNA impaired the insulin-induced adipogenic differentiation. Histamine receptors and histamine-like biogenic amines may play a role in inducing adipogenesis in response to insulin.