The Herpes Simplex Virion Protein 16 (VP16) activates transcription through a series of protein/protein interactions involving its highly acidic transactivation domain (TAD). The acidic TAD of VP16 (VP16TAD) has been shown to interact with several partner proteins both in vitro and in vivo, and many of these VP16 partners also bind the acidic TAD of the mammalian tumor suppressor protein p53. For example, the TADs of VP16 and p53 (p53TAD) both interact directly with the p62/Tfb1 (human/yeast) subunit of TFIIH, and this interaction correlates with their ability to activate both the initiation and elongation phase of transcription. In this manuscript, we use NMR spectroscopy, isothermal titration calorimetery (ITC) and site-directed mutagenesis studies to characterize the interaction between the VP16TAD and Tfb1. We identify a region within the carboxyl-terminal subdomain of the VP16TAD (VP16C) that has sequence similarity with p53TAD2 and binds Tfb1 with nanomolar affinity. We determine an NMR structure of a Tfb1/VP16C complex, which represents the first high-resolution structure of the VP16TAD in complex with a target protein. The structure demonstrates that like p53TAD2, VP16C forms a 9-residue alpha-helix in complex with Tfb1. Comparison of the VP16/Tfb1and p53/Tfb1 structures clearly demonstrates how the viral activator VP16C and p53TAD2 shares numerous aspects of binding to Tfb1. Despite the similarities, important differences are observed between the p53TAD2/Tfb1 and VP16C/Tfb1 complexes, and these differences demonstrate how selected activators such as p53 depend on phosphorylation events to selectively regulate transcription.

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The general transcription factor IIH is recruited to the transcription preinitiation complex through an interaction between its p62/Tfb1 subunit and the ␣-subunit of the general transcription factor IIE (TFIIE␣). We have determined that the acidic carboxyl terminus of TFIIE␣ (TFIIE␣336-439) directly binds the amino-terminal PH domain of p62/Tfb1 with nanomolar affinity. NMR mapping and mutagenesis studies demonstrate that the TFIIE␣ binding site on p62/Tfb1 is identical to the binding site for the second transactivation domain of p53 (p53 TAD2). In addition, we demonstrate that TFIIE␣336-439 is capable of competing with p53 for a common binding site on p62/ Tfb1 and that TFIIE␣336-439 and the diphosphorylated form (pS46/ pT55) of p53 TAD2 have similar binding constants. NMR structural studies reveal that TFIIE␣336-439 contains a small domain (residues 395-433) folded in a novel ␤␤␣␣␣ topology. NMR mapping studies demonstrate that two unstructured regions (residues 377-393 and residues 433-439) located on either side of the folded domain appear to be required for TFIIE␣336-439 binding to p62/Tfb1 and that these two unstructured regions are held close to each other in threedimensional space by the novel structured domain. We also demonstrate that, like p53, TFIIE␣336-439 can activate transcription in vivo. These results point to an important interplay between the general transcription factor TFIIE␣ and the tumor suppressor protein p53 in regulating transcriptional activation that may be modulated by the phosphorylation status of p53.NMR ͉ phosphatidylinositol 5-phosphate ͉ transcription regulation ͉ activation domains ͉ isothermal titration calorimetry I n eukaryotic cells, a crucial event for the transcription cycle of protein-coding genes is the assembly of RNA polymerase II (RNAP II) and the general transcription factors at the DNA promoter region to form the preinitiation complex (PIC) (1, 2). According to the sequential assembly model of the PIC formation, the last steps in transcription initiation are the binding of the general transcription factor IIE (TFIIE) to the PIC, followed by the TFIIE-assisted recruitment of the general transcription factor IIH (TFIIH) (3, 4). The association of TFIIE and TFIIH to the initial PIC is essential for RNAP II to proceed from an unphosphorylated form to a phosphorylated form and also for the transition from the initiation to the elongation phase of transcription (5, 6).TFIIE is composed of two subunits, ␣ and ␤. The larger ␣-subunit (TFIIE␣) contains several functional domains that are mainly located at the amino-terminal half of the protein. These domains have been demonstrated to be critical for basal transcription and cell growth (7, 8), and they are required for the interaction of TFIIE␣ with the ␤-subunit of TFIIE (TFIIE␤), RNAP II, and other transcription factors, as well as for regulating the enzymatic activities of TFIIH (4, 7, 9). TFIIE␤ also possesses functional domains that are responsible for important interactions with TFIIE␣, RNAP II, TFIIB, and TFIIF (10).H...

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Malfunctions in transcriptional regulation are associated with a number of critical human diseases. As a result, there is considerable interest in designing artificial transcription activators (ATAs) that specifically control genes linked to human diseases. Like native transcriptional activator proteins, an ATA must minimally contain a DNA-binding domain (DBD) and a transactivation domain (TAD) and, although there are several reliable methods for designing artificial DBDs, designing artificial TADs has proven difficult. In this manuscript, we present a structure-based strategy for designing short peptides containing natural amino acids that function as artificial TADs. Using a segment of the TAD of p53 as the scaffolding, modifications are introduced to increase the helical propensity of the peptides. The most active artificial TAD, termed E-Cap-(LL), is a 13-mer peptide that contains four key residues from p53, an N-capping motif and a dileucine hydrophobic bridge. In vitro analysis demonstrates that E-Cap-(LL) interacts with several known p53 target proteins, while in vivo studies in a yeast model system show that it is a 20-fold more potent transcriptional activator than the native p53-13 peptide. These results demonstrate that structure-based design represents a promising approach for developing artificial TADs that can be combined with artificial DBDs to create potent and specific ATAs.

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1 Endothelin-1 (ET-1) is a bicyclic 21-amino-acid peptide causing a potent and sustained vasoconstriction, mainly through the ET A receptor subtype. So far, no selective ET A agonists are described in the literature. 2 A series of truncated and chemically modified ET-1 analogues were obtained through solid-phase peptide synthesis and their biological activity was assessed on rat thoracic aorta rings (ET A receptors) and guinea-pig lung parenchyma strips (ET B receptors). 3 Structure -activity studies led to the identification of ET-1 fragments exhibiting an ET A selective agonistic activity. 21 ]ET-1(9 -21), showed a selective ET A activity (EC 50 : 3.0 Â 10 À6 M). None of the numerous analogues of the series exhibited substantial effects in the guinea-pig lung parenchyma bioassay. 5 Thus, this study describes the first compounds showing a significant bioactivity in an ET A pharmacological preparation while being inactive in an ET B paradigm. They show that the ET-1 pharmacophores, responsible for the ET A -mediated actions, are located within the 9 -21 segment of the molecule. Moreover, the bicyclic structure of ET-1 does not appear as essential for the ET A -related vasoconstriction. Results also suggest that the positive charge of the Lys 9 side chain participates in an intramolecular ionic bond with the carboxylate function of Asp 18 .

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