PhD Thesis Ex-vivo and in-silico Study of p53 Transactivation, Dynamics in Budding Yeast


TP53 gene codes for protein p53 which is possibly the most important tumor suppressor. This activity of p53 is triggered when the cell experiences harmful stress like ultra violet (UV) radiation, depleted oxygen levels (Hypoxia) etc. Any biological process is a series of coordinated controlled events that forms a complex interactive inter-regulatory network. These series of co-ordinations and controls can be both physical (binding/unbinding events) and non-physical interactions (gene and protein regulatory networks). In the case of p53, its manifestations observed are a combination of the above. To analyze the p53 activity at a simpler level, we have tried to minimize the non-physical component by removing the known gene and protein regulatory modes, thereby allowing us to study the protein inside a simple model organism, budding yeast. This gives us an option to hypothesize, design, quantify, test and validate the activity of p53 by in-silico techniques of systems biology. Wild type (wt) and mutant p53s which differ in the capacity to form protein-protein complexes (oligomerization) are prepared. Both these protein forms binding on p21-5’ natural response element (RE) and its variants were studied. For this, specialized gene reporter systems with Gal promoter-TP53gene-luciferase reporter were designed and incorporated into the budding yeast cells. Galactose sugar at varying concentrations was administered to these cells, which eventually induce p53 production and the subsequent reactions (formation of dimer of dimers on the DNA) follow. Mathematical models with known binding and unbinding scenarios were prepared with the aim to understand if the dimers occupying the specific DNA (RE) help each other (cooperative) or not. The two mathematical models were used to fit the experimental data, and the binding/unbinding rates were estimated. We observed that the estimated values for one of the steps in the binding/unbinding were different from parameters of preceding steps. From the estimated values of binding/unbinding for mutant p53, it was observed that their DNA binding domains incorporate cooperativity in spite of the absence of tetramerization domain. Cooperativity was more conspicuous from the estimated parameters for mutant p53 than that of wild type p53. Similar experiments and fitting using both models were repeated with different topologies of REs. Cooperativity between the dimers on RE seems to depend on multiple factors like whether the protein is mutated and whether there is spacer between the binding sites of RE.

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S. Muppirisetty