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The mdm2 gene product is an important regulator of p53 function and stability. mdm2 is an E3 ubiquitin ligase for p53 and the RING finger domain of mdm2 is critical for ligase activity. Ubiquitin (Ub) conjugation is a general targeting modification and poly-ubiquitin chains specifically target proteins to the proteasome for degradation. In this report, we show that the multistep cascade of mdm2-mediated p53 ubiquitination can be reduced to three purified recombinant proteins: ubiquitin-conjugated E2, mdm2, and p53. This simplification allows enzymatic analysis of the isolated ligase reaction. The simplified reaction recapitulates the ubiquitination of p53 observed with individual components and the p53-Ub (n) is qualitatively similar to p53-Ub (n) detected in lactacystin-treated cells. Surprisingly, we find that p53 is modified with multiple mono-ubiquitin moieties as opposed to a poly-ubiquitin chain. Finally, kinetic analysis indicates the transfer reaction proceeds either through a modified Ping Pong mechanism involving requisite enzyme isomerization steps, or through a Rapid Equilibrium Random Bi Bi mechanism involving very large anti-cooperative interactions between the two substrate binding pockets on the enzyme, mediated through allosteric changes in enzyme structure.The p53 gene product is an important tumor suppressor and is inactivated by deletion or mutation in approximately 50% of all human cancer. The p53 protein functions as a transcription factor that binds DNA and induces the expression of a number of genes involved in cell growth arrest, DNA repair, and apoptosis. p53 is maintained at low steady-state levels in the cell and is induced and activated post-translationally by various signaling pathways that respond to cellular stress (1, 2). Cellular insults that initiate the stress response and activate p53 include DNA-damaging agents (chemical, UV, and ionizing radiation), oxygen stress, or the inappropriate activation of oncogenes. Activated p53 induces either growth arrest or apoptosis depending on the extent and duration of signals generated from the damage (3, 4). The post-translational modifications involved in p53 activation and increased steady-state levels of the protein include phosphorylation, dephosphorylation, acetylation, sumoylation, and ubiquitination. The stability and half-life of p53 are tightly regulated by mdm2 and the ubiquitin-proteasome pathway (5-7). Recent evidence suggests that mdm2 is an E3 1 ubiquitin ligase for p53 (8, 9). A number of critical regulatory proteins in the cell are modified by ubiquitin (Ub) conjugation. Proteasomal degradation of key regulatory proteins control biological events involving the cell cycle, differentiation, immune responses, DNA repair, chromatin structure, and apoptosis (10). The initial step in the Ub cascade is the activation of Ub by the ubiquitin-activating enzyme (E1). E1 hydrolyzes ATP to AMP and pyrophosphate to generate a thioester bond between the active site Cys of E1 and the carboxylterminal Gly of Ub. The activated Ub is transf...

A class of regulators of eukaryotic gene expression contains a conserved amino acid sequence responsible for protein oligomerization and binding to DNA. This structure consists of an arginine- and lysine-rich basic region followed by a helix-loop-helix motif, which together mediate specific binding to DNA. Peptides were prepared that span this motif in the MyoD protein; in solution, they formed alpha-helical dimers and tetramers. They bound to DNA as dimers and their alpha-helical content increased on binding. Parallel and antiparallel four-helix models of the DNA-bound dimer were constructed. Peptides containing disulfide bonds were engineered to test the correctness of the two models. A disulfide that is compatible with the parallel model promotes specific interaction with DNA, whereas a disulfide compatible with the antiparallel model abolishes specific binding. Electron paramagnetic resonance (EPR) measurements of nitroxide-labeled peptides provided intersubunit distance measurements that also supported the parallel model.