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Identification of an additional negative regulatory region for p53 sequence-specific DNA binding
B F Müller-Tiemann1, T D Halazonetis, J J Elting
1Institute for Bone and Joint Disorders and Cancer, Bayer Corporation, Pharmaceutical Division, 400 Morgan Lane, West Haven, CT 06516, USA. beate.muellertiemann@schering.de
Abstract:
The DNA binding activity of p53 is crucial for its tumor suppressor function and is subject to tight regulation. Previous studies revealed that the inhibitory function of the p53 C terminus is implicated in the latent, low affinity sequence-specific DNA binding activity of p53 in the uninduced state. Sequence-specific DNA binding of p53 has been shown to be activated by several posttranslational modifications and interacting proteins that target predominantly the C terminus. Moreover, several authors have shown that synthetic peptides corresponding to p53 C-terminal sequences activate p53 sequence-specific DNA binding. In an effort to identify the interaction site of p53 with these activating peptides we assessed complex formation between p53 deletion constructs and C-terminal activating peptides by peptide affinity precipitation. This study revealed that two distal regions of the p53 molecule contribute synergistically to the interaction with activating C-terminal peptides: amino acids 80-93 and 364-393. The C-terminal residues 364-393 are already well characterized as having negative regulatory function. DNA binding analyses with these deletion constructs reveal a comparable negative regulatory activity for residues 80-93, defining this region as a previously unidentified negative regulatory domain of p53. Furthermore, synthetic peptides spanning this newly identified proline-rich negative regulatory region (residues 80-93) are able to activate p53 sequence-specific DNA binding in vitro. We suggest that both negative regulatory regions, residues 80-93 and 364-393, contribute cooperatively to the maintenance of the latent, low-affinity DNA binding conformation of p53.
Insights
Researchers identified a new negative regulatory domain in the p53 protein (amino acids 80-93). This domain, along with a known region, cooperatively maintains p53
Area of Science:
- Molecular Biology
- Protein Regulation
- Cancer Research
Background:
- The tumor suppressor protein p53's DNA binding is tightly regulated.
- The C-terminus of p53 is known to inhibit its DNA binding activity in an uninduced state.
- Posttranslational modifications and interacting proteins activate p53 DNA binding, primarily by targeting the C-terminus.
Purpose of the Study:
- To identify the specific interaction sites of p53 with activating peptides.
- To characterize previously unidentified regulatory regions of p53 involved in DNA binding.
Main Methods:
- Peptide affinity precipitation assays using p53 deletion constructs and C-terminal activating peptides.
- DNA binding analyses of p53 deletion constructs.
- In vitro activation of p53 DNA binding using synthetic peptides.
Main Results:
- Two distal regions of p53 (amino acids 80-93 and 364-393) synergistically interact with activating peptides.
- Residues 80-93 were identified as a novel negative regulatory domain of p53, exhibiting similar inhibitory function to residues 364-393.
- Synthetic peptides from the 80-93 region activated p53 sequence-specific DNA binding in vitro.
Conclusions:
- The p53 regions 80-93 and 364-393 cooperatively maintain p53 in a latent, low-affinity DNA binding conformation.
- The newly identified domain (80-93) provides new insights into the regulation of p53 tumor suppressor activity.