Single-molecule Studies Of p53 Sliding Along DNA

Abstract

To locate its target site on DNA, a transcription factor (TF) must recognize its site amongst millions to billions of alternative sites on DNA. Studies suggested that TFs in order to facilitate their search process alternate between 3D diffusion in solution and 1D diffusion along DNA. The duration of such a search depends on the rate at which a TF slides along DNA and the frequency with which it alternates between 1D and 3D diffusion.We are interested in the 1D searching mechanism of p53, a transcription factor that functions as a tumor suppressor in human cells. We are using single-molecule techniques to observe diffusion of the fluorescently labeled p53 proteins along individual, stretched DNA molecules. In our previous studies, we determined the 1D diffusion coefficient of p53 protein. By measuring the 1D diffusion of the p53 protein as a function of ionic strength, we determined that the p53 protein maintains close contact with the DNA duplex and tracks the helical pitch. Current work involves the characterization of the role of the different protein domains in sliding. The C-terminus of p53 is suggested to be responsible for keeping the protein in contact with DNA by non-specifically interacting with the negatively charged backbone of DNA, while the core domain is suggested to be responsible for specifically binding the target site. We will present single-molecule data on the diffusional mobility along DNA of the C-terminal domain of p53, the p53 lacking its C-terminus, and the core domain of p53.