p53 requires an intact C-terminal domain for DNA binding and transactivation

The tumor suppressor p53 plays a critical role in mediating cellular response to a wide range of environmental stresses. p53 regulates these processes mainly by acting as a short-lived DNA binding protein that stimulates transcription from numerous genes involved in cell cycle arrest, programmed cell death, and other processes. To investigate the importance of the C-terminal domain of p53, we generated a series of deletion and point mutations in this region and analyzed their effects on p53 transcription activity. Our results show that C-terminal deletion and point mutations at K320 and K382 abolish p53-mediated transcription in the context of DNA or chromatin. This defect is specific for DNA molecules because inactive mutants fail to bind a consensus p53 response element in both free DNA and nucleosomes. Chromatin immunoprecipitation assays further substantiate the importance of the p53 C-terminal domain for the targeted localization of p53 and the concomitant recruitment of p300 onto p53-responsive genes. Moreover, a synthetic peptide comprising the last 30 amino acids of p53 interacts with the N-terminal and C-terminal domains of p53 and antagonizes p53-dependent transcription. Taken together, our data reveal a functional requirement for the p53 C-terminal domain in p53 transactivation and support a working model in which the C-terminus serves as a positive regulator for N-terminal activation and central DNA binding domains.