TRANSACTIVATION ABILITY OF P53 TRANSCRIPTIONAL ACTIVATION DOMAIN IS DIRECTLY RELATED TO THE BINDING-AFFINITY TO TATA-BINDING PROTEIN

Tumor suppressor protein p53 is a potent transcriptional activator and regulates cell growth negatively. To characterize the transcriptional activation domain (TAD) of p53, various point mutants were constructe d in the context of Ga14 DNA binding domain and tested for their trans activation ability. Our results demonstrated that the positionally con served hydrophobic residues shared with herpes simplex virus VP16 and other transactivators are essential for transactivation. Also, the neg atively charged residues and proline residues are necessary for full a ctivity, but not essential for the activity of p53 TAD. Deletion analy ses showed that p53 TAD can be divided into two subdomains, amino acid s 1-40 and 43-73. An in vitro glutathione S-transferase pull-down assa y establishes a linear correlation between p53 TAD mediated transactiv ation in vivo and the binding activity of p53 TAD to TATA-binding prot ein (TBP) in vitro. Mutations that diminish the transactivation abilit y of Ga14-p53 TAD also impair the binding activity to TBP severely, Ou r results suggest that at least TBP is a direct target for p53 TAD and that the binding strength of TAD to TBP (TFIID) is an important param eter controlling activity of p53 TAD. In addition, circular dichroism spectroscopy has shown that p53 TAD peptide lacks any regular secondar y structure in solution and that there is no significant difference be tween the spectra of the wild type TAD and that of the transactivation -deficient mutant type.