Use of the two-hybrid system to identify the domain of p53 involved in oligomerization.
We used a yeast-based genetic assay, the two-hybrid system, to characterize the domain of the tumor-suppressor p53 involved in oligomerization. This assay relies on the reconstitution of the function of a transcriptional activator, the yeast GAL4 protein, via the interaction of a protein fused to the DNA-binding domain of GAL4 with a protein fused to the transcriptional activation domain of GAL4. We show by a reconstruction experiment that this approach could detect the interaction of p53 deleted for its N-terminal activation domain with SV40 large T antigen. We then searched a library of human proteins present as activation domain hybrids for proteins that can interact with the hybrid of p53 with the DNA-binding domain. This search identified 36 plasmids containing the p53 gene, representing 10 different classes. These results provide an additional in vivo demonstration of p53 oligomerization. The smallest p53 fragment identified from screening the library contained only amino acids 331-393, indicating that this small C-terminal fragment is sufficient to mediate oligomerization. In addition, a mutant p53 protein could bind to the wild-type protein in this assay, providing support for the idea that mutant forms of p53 act in a dominant-negative manner through C-terminal oligomerization with the wild type.
Department of Microbiology, State University of New York, Stony Brook 11794.
K
K
Iwabuchi
B
B
Li
P
P
Bartel
S
S
Fields
D000952:Antigens, Polyomavirus Transforming / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D015723:Gene Library; D016158:Genes, p53; D006801:Humans; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D016159:Tumor Suppressor Protein p53 / Q000235:genetics* / Q000378:metabolism*; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D000952:Antigens, Polyomavirus Transforming; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D016159:Tumor Suppressor Protein p53; D001616:beta-Galactosidase