Four p53 DNA-binding domain peptides bind natural p53-response elements and bend the DNA.
Recent structural studies of the minimal core DNA-binding domain of p53 (p53DBD) complexed to a single consensus pentamer sequence and of the isolated p53 tetramerization domain have provided valuable insights into their functions, but many questions about their interacting roles and synergism remain unanswered. To better understand these relationships, we have examined the binding of the p53DBD to two biologically important full-response elements (the WAF1 and ribosomal gene cluster sites) by using DNA circularization and analytical ultracentrifugation. We show that the p53DBD binds DNA strongly and cooperatively with p53DBD to DNA binding stoichiometries of 4:1. For the WAF1 element, the mean apparent Kd is (8.3 +/- 1.4) x 10(-8) M, and no intermediate species of lower stoichiometries can be detected. We show further that complex formation induces an axial bend of at least 60 degrees in both response elements. These results, taken collectively, demonstrate that p53DBD possesses the ability to direct the formation of a tight nucleoprotein complex having the same 4:1 DNA-binding stoichiometry as wild-type p53 which is accompanied by a substantial conformational change in the response-element DNA. This suggests that the p53DBD may play a role in the tetramerization function of p53. A possible role in this regard is proposed.
Balagurumoorthy
P
P
Department of Biochemistry, University of Nevada Reno 89557-0014, USA.
Sakamoto
H
H
Lewis
M S
MS
Zambrano
N
N
Clore
G M
GM
Gronenborn
A M
AM
Appella
E
E
Harrington
R E
RE
D001483:Base Sequence; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000235:genetics; D004247:DNA / Q000378:metabolism*; D004275:DNA, Ribosomal / Q000235:genetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010446:Peptide Fragments / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D012045:Regulatory Sequences, Nucleic Acid; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D014461:Ultracentrifugation
D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004275:DNA, Ribosomal; D010446:Peptide Fragments; D016159:Tumor Suppressor Protein p53; D004247:DNA