UC San Diego

The biochemical mechanisms of transcription specificity in the p53 family and NFkappaB family of transcription factors in the context of DNA sequence recognition are not well-defined. Given the loose consensus of the DNA binding sites recognized by the p53 and NFkappaB family, we investigated the structural and biochemical contributions to DNA sequence recognition. In chapter 2, we show the effects of perturbing a loop feature in the DNA-binding domain of p73 on cooperative binding that is selective for different DNA sequences using x-ray crystallography and fluorescence polarization. This is the first structural evidence of a L1 loop-dependent mechanism of sequence recognition that implicates the role of L1 loop acetylation to selective activation of apoptotic genes. In chapter 3, we continued to investigate the structural mechanism of recognition by crystallizing wild-type p73 DNA-binding domain with a native p53 DNA response element, which showed conserved features of DNA recognition. In the final chapter 4, the effects of mutating a native NFkappaB DNA response element were investigated with RelA using mammalian luciferase assay, electrophoretic mobility shift assay, and in vitro chromatin transcription assay to corroborate the “ruler” mechanism of recognition observed in crystal structures. The RelA transactivation domain was shown to contribute to sequence specificity that has not been shown before. Together, these studies show structural features that are important in sequence recognition and further refine mechanisms of transcription specificity based on cooperative, multi-domain interactions.