Human tumor-derived p53 proteins exhibit binding site selectivity and temperature sensitivity for transactivation in a yeast-based assay

Abstract

p53 is a sequence-specific transcriptional activator with a number of known target genes which contain p53-responsive elements. Mutations in p53 have been identified within its sequence-specific DNA binding domain in more than half of all human tumors, although a subset of tumor-derived p53 mutants have retained the ability to bind DNA and activate transcription under certain conditions. In order to broaden our understanding of this transactivating ability, we examined the efficacy by which p53 mutants bind to and activate reporters in an Saccharomyces cerevisiae-based assay. Analysis of 19 human tumor-derived p53 mutants, spanning the DNA binding domain of p53 and including the 'hot-spot' class, revealed a broad array of transcriptional transactivation abilities at 24 degrees C, 30 degrees C and 37 degrees C, despite the fact that each mutant had originally been identified as being inactive for transactivation in yeast against a single p53-responsive RGC site-containing reporter. One class of mutants (P177L, R267W, C277Y and R283H) retained wild-type or near wild-type activity that is binding site-selective, even at physiological temperature (37 degrees C). Another class of mutants (V143A, M1601/A161T, H193R, Y220C and 1254F), all positioned for maintaining the beta-scaffold of p53, also retained selective activity, but preferentially at sub-physiological temperatures (24 degrees and 30 degrees C). Strikingly, however, in contrast to the other tumor derived mutants, all of the previously identified 'hot-spot' mutants were completely inactive with all sites tested. Moreover, a double mutant, L22E/W23S, located within the activation region and previously shown to be transcriptionally inactive in fibroblasts, retained wild-type or near wild-type binding site-selective activity in yeast. Finally, we found that transcriptional activity in vivo does not necessarily correlate with DNA binding in vitro.