Low-level p53 expression changes transactivation rules and reveals superactivating sequences.

Transcriptional activation by the tumor suppressor p53 is considered to depend on cellular level, although there are few systems where this dependence on cellular level of p53 has been directly addressed. Previously, we reported that transactivation from p53 targets was sensitive to both p53 amount and DNA sequence, with some sequences being responsive to much lower p53 levels than others when examined in yeast model systems or human cells. Because p53 is normally present at low levels and perturbations might lead to small increases, we examined transactivation under limiting p53. Unlike the positive relationship between transactivation and binding affinity from target sequences at high cellular levels of human p53 in yeast, no such relationship was found at low levels. However, transactivation in the yeast system and the torsional flexibility of target sequences were highly correlated, revealing a unique structural relationship between transcriptional function and sequence. Surprisingly, a few sequences supported high transactivation at low p53 levels in yeast or when transfected into human cells. On the basis of kinetic and flexibility analyses the "supertransactivation" property was due to low binding off rates of flexible target sites. Interestingly, a supertransactivation response element can differentiate transcriptional capacities of many breast cancer-associated p53 mutants. Overall, these studies, which are relevant to other transcription factors, address the extent to which transactivation properties of p53 target sequences are determined by their intrinsic physical properties and reveal unique rules of engagement of target sequences at low p53 levels.

Sequence-dependent cooperative binding of p53 to DNA targets and its relationship to the structural properties of the DNA targets.

The prime mechanism by which p53 acts as a tumor suppressor is as a transcription factor regulating the expression of diverse downstream genes. The DNA-binding domain of p53 (p53DBD) interacts with defined DNA sites and is the main target for mutations in human primary tumors. Here, we show that the CWWG motif, found in the center of each consensus p53 half-site, is a key player in p53/DNA interactions. Gel-mobility-shift assays provide a unique opportunity to directly observe the various oligomeric complexes formed between p53DBD and its target sites. We demonstrate that p53DBD binds to p53 consensus sites containing CATG with relatively low cooperativity, as both dimers and tetramers, and with even lower cooperativity to such sites containing spacer sequences. p53DBD binds to sites containing CAAG and CTAG with measurable affinity only when imbedded in two contiguous p53 half-sites and only as tetramers (with very high cooperativity). There are three orders-of-magnitude difference in the cooperativity of interaction between sites differing in their non-contacted step, and further two orders-of-magnitude difference as a function of spacer sequences. By experimentally measuring the global structural properties of these sites, by cyclization kinetics of DNA minicircles, we correlate these differences with the torsional flexibility of the binding sites.