טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentAmar Keren Or
SubjectThe Influence of the Sequences Flanking the TATA-Box on TATA
Binding Protein/TATA Box Interactions
DepartmentDepartment of Biology
Supervisor Professor Haran Tali


Abstract

The TATA box binding protein (TBP) recognizes its target sites on double stranded DNA, the TATA box, via mechanism that is based on the recognition of the local conformation on the DNA double helix (“indirect readout”). Although the crystal structure of the TBP/TATA box complex shows that TBP binds only to the core TATA box, there is evidence from our previous studies that sequences flanking the core TATA box exert an influence on the interaction of TBP with the TATA box. To further study this influence, I used a variety of flanking sequences and measured the stability of the resulting TBP/TATA box complexes. My study suggests that for stable TBP/TATA box complexes the preferred sequences flanking the TATA box are those with intermediate isotropic flexibility. Moreover, I studied systematically the nature of the flanking sequences forming stable complexes with TBP by conducting an “in vitro selection” experiments, to select the most stable complexes from a pool of two TATA boxes targets (E4 and MLP) each with ten random sequences in the region flanking those TATA boxes. This study is in progress and currently I can state only that with the E4 target it appears that the fraction of bound molecules reached a plateau, whereas with wtMLP target it keeps growing up constantly. This can be explained by the more stringent demands on the E4 target for certain flanking sequences. To complete the picture, I searched the eukaryotic database (EPD) for natural sequences flanking TATA boxes. My search suggests that there is a correlation between the results of dissociation kinetics experiments and the frequency of occurrence of sequences flanking the TATA-box. Further I used methylated cytosine in the flanking sequences in order to establish the mechanism by which the flanking sequences influence the binding of TBP to its target sites. My findings support a structural influence model.