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Sequence-Dependent Kinetics of One-Dimensional Diffusion of p53 on DNA

2009; Elsevier BV; Volume: 96; Issue: 3 Linguagem: Inglês

10.1016/j.bpj.2008.12.2125

1542-0086

Jason S. Leith, Anahita Tafvizi, Fang Huang, Alan R. Fersht, Leonid A. Mirny, Antoine M. van Oijen,

RNA and protein synthesis mechanisms

Proteins such as transcription factors that must locate and bind to one or a small number of cognate sites on the genome have been suggested to undergo alternating rounds of one-dimensional (1D) and three-dimensional (3D) diffusion. It has been argued that a single protein-DNA energy landscape as a function of the position of the protein on DNA cannot be both smooth enough for a rapid search process and rugged enough so that the protein binds stably to its cognate site (Slutsky and Mirny, Biophys. J. 87 (2004) 4021). We have offered instead a model of kinetic pre-selection whereby proteins diffuse with low friction along a search landscape and pause on cognate sites before adopting transitioning to a recognition landscape which accounts for the rapid localization process observed experimentally (submitted). The transcription factor p53's role in tumor suppression gives it a biological need to locate and bind its promoters quickly. We developed a single-molecule fluorescence imaging approach to directly observe the protein diffusing in 1D on stretched DNA in vitro (Tafvizi et al., Biophys. J. 95 (2008) L01). By using total internal reflection microscopy to image fluorescently labeled p53 diffusing on stretched duplex DNA, we obtain information on the protein's diffusional properties as a function of its position on the DNA. Using a bioinformatics approach, we have identified a number of sites on our substrate DNA that closely resemble known p53 promoters. We present initial results that demonstrate the feasibility of this approach to correlate high-resolution information on diffusional properties with the positions of these binding sites.