טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentSenitzki Alon
SubjectStructural Properties of Natural p53 Target Genes Binding
Sites
DepartmentDepartment of Biology
Supervisor Professor Haran Tali
Full Thesis textFull thesis text - English Version


Abstract

The p53 protein is a transcription factor that regulates the expression of more than one hundred genes in the human genome. The main function of p53 is tumor suppression. This function is achieved by inducing the transcription of two main groups of target genes. The first group includes genes that promote permanent or temporary cell-cycle arrest. The second group includes genes that promote an apoptotic response. p53-dependent transactivation of a target gene always involves its binding to a DNA response-element (RE). The consensus sequence of p53 RE is highly degenerate and consists of two decameric half-sites with the motif RRRCWWGGG (R = A, G; W = A, T; Y = C, T) separated by a variable number of base-pairs. The way by which p53 is able to differentially regulates target genes that promote different cellular outcomes is unclear. The mechanisms by which p53 interacts with its REs involves both direct and indirect readout. It was recently demonstrated that torsional flexibility of p53 REs influences its binding affinity and binding cooperativity, and at low cellular p53 concentration it correlates with transactivation capacity.

            The aim of this work was to study the structure of naturals p53 REs. Three validated p53 REs were analyzed by the method of cyclization kinetics: the distal and proximal REs on the p21 promoter, referred to as p21-5' and p21-3', and a RE on the Bax promoter, referred to as BaxB. The cyclization kinetics assay allowed the quantification of global aspects of DNA structure and dynamics. Here I used cyclization kinetics to characterize six half-sites of three natural p53 REs by four structural parameters: bend, bend flexibility, twist and twist flexibility. In addition, I used the electrophoretic mobility shift assay to determine binding affinity and cooperativiy of p53 to three variants of the p21-5' RE.

            I showed that natural p53 REs are relatively straight, and are similar to each other with regard to their bending flexibility. In contrast, p53 REs display a wide range of torsional flexibility values. Moreover, natural p53 REs have an asymmetric torsional flexibility, with the two half-sites comprising each natural p53 RE displaying a considerable difference in this parameter. In addition, I showed that asymmetric torsional flexibility influence p53 binding cooperativity.

            We suggests that torsional flexibility of p53 REs, together with p53 cellular concentration, is a major contributor to the ability of p53 to differentially regulates the expression of it functionally diverse target genes.