p53-response elements (p53-REs) are organized as two repeats of a palindromic DNA segment spaced by 0 to 20 base pairs (bp). Several experiments indicate that in the vast majority of the human p53-REs there are no spacers between the two repeats; those with spacers, particularly with sizes beyond two nucleotides, are rare. This raises the question of what it indicates about the factors determining the p53-RE genomic organization. Clearly, given the double helical DNA conformation, the orientation of two p53 core domain dimers with respect to each other will vary depending on the spacer size: a small spacer of 0 to 2 bps will lead to the closest p53 dimer-dimer orientation; a 10-bp spacer will locate the p53 dimers on the same DNA face but necessitate DNA looping; while a 5-bp spacer will position the p53 dimers on opposite DNA faces. Here, via conformational analysis we show that when there are 0–2 bp spacers, p53-DNA binding is cooperative; however, cooperativity is greatly diminished when there are spacers with sizes beyond 2 bp. Cooperative binding is broadly recognized to be crucial for biological processes, including transcriptional regulation. Our results clearly indicate that cooperativity of the p53-DNA association dominates the genomic organization of the p53-REs, raising questions of the structural organization and functional roles of p53-REs with larger spacers. We further propose that a dynamic landscape scenario of p53 and p53-REs can better explain the selectivity of the degenerate p53-REs. Our conclusions bear on the evolutionary preference of the p53-RE organization and as such, are expected to have broad implications to other multimeric transcription factor response element organization.

p53-response elements (p53-REs) are 20 base pair DNA segments embedded in the genome that are able to bind anti-tumor protein p53 and trigger biological functions such as DNA repair or self-destruction of the cell. These functions are modulated through selective binding of p53 to degenerate p53-REs. Understanding how the cells choose p53-REs for enacting a specific biological function is crucial for obtaining insight into cancer development. Experimental data indicate that the majority of p53-REs contain a small intervening spacer in the middle of their p53-REs. Here, we propose that there is a relationship between the organization of the p53-REs and binding cooperativity. To test this hypothesis, analysis of existing crystal structures and modeling of p53-DNA complexes was undertaken. The outcome shows that when there are 0–2 base pair spacers, there are more interactions between the p53 subunits, and the p53-DNA binding is cooperative. When the spacer sizes are larger, the interactions among p53 subunits are diminished. Our results indicate that cooperativity of the p53-DNA association dominates the genomic organization of the p53-REs and suggest that different mechanisms of activation may be at play for those p53-REs with large spacers.