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Structural Discrimination between Activating and Repressing Response Elements in the p53 Protein Family

Abstract

The differential expression of genes is a fundamental hallmark of cell development. Transcription factors are key proteins that trigger gene transcription. Traditionally, it is considered that a transcription factor binding to a response element is sufficient to trigger transactivation of the regulated gene. Nonetheless, a view is emerging that considers that sole binding of the transcription site to a response element does not warrant gene activation. In my thesis work, I study by structural and biochemical techniques how the members of the p53 transcription family distinguish between response elements that will trigger gene activation or gene repression. The p53 transcription family that comprises p53, p63 and p73 are some of the most studied proteins, particularly, because mutations in the p53 gene are present in more than 50% of cancers. Together, the three transcription factors regulate hundreds of genes in development and stress pathways. To trigger transcription, the members of the p53 family bind in a specific manner to a response element, a DNA sequence composed of two 10 bp half-sites response elements with the 5'-PuPuPuCA/TGPyPyPy-3' consensus sequence. Functional p53 and p73 require the formation of oligomers to bind with high affinity and specificity to its response element. Recently, based on functional studies, response elements have been divided into activating and repressing. Although, there is vast amount of information about p53 and p73 function and structure, little is known about their molecular mechanism to discriminate between activating and repressing response elements. By using DNA binding assays with fluorescence polarization and crystal structure determination of protein-DNA complexes, I studied biochemically and structurally how p53 and p73 are able to distinguish between activating and repressing response elements. The analysis of my data allowed me to conclude that : 1) the different nucleotides in the p53-p73 half-site response element have each a distinctive role in discriminating whether the DNA-binding domain will distinguish between strongly or weakly bound response elements; 2) the DNA binding domain dimer changes conformation to discriminate between activating and repressing response elements in a mechanism triggered by a lysine in loop L1 and that involves the switch of the central adenine to a Hoogsteen conformation; and 3) the oligomerization domain increases cooperativity and specificity, while the acetylation of the lysine in loop L1 does not have an effect on the binding affinity of the DNA binding domain to its response element. My work represents the clearest molecular view to date on how a transcription factor binding to its response element is not sufficient to acquire a conformation that promotes transactivation

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