טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentRoles Asia
SubjectCan Tryptophan Repressor Interact with its Target Site
Exclusively through Structural Readout?
DepartmentDepartment of Biology
Supervisor Professor Tali Haran


Abstract

Our understanding of sequence specific protein-DNA interactions evolved over the last thirty years with the development of the methods available to study these interactions. It was first believed that the protein recognizes its target site by direct contact with the side chains of the protein and the hydrogen donor and the acceptor groups on the DNA bases in the major groove of the double helix. This mode of interaction was termed direct readout. The analysis of the crystal structure of the trp repressor/operator complex revealed only a few direct hydrogen bonds in the direct mode of interaction. However, there were specific interactions of the protein side chains to the donor and acceptor groups of the DNA bases mediated through water molecules. This mode of interaction was termed indirect readout.
Our current view of specific protein-DNA interactions is a combination of direct readout, indirect readout and structural readout. Structural readout is the recognition of the structural parameters of the double helix as well as its deformations from standard B-DNA structure. The relative contribution of each mode of recognition to the formation and stability of the complex varies largely from one complex to another. It is currently believed that protein-DNA interactions are based primarily on direct and indirect readout, whereas structural recognition has only a supportive role in this interaction.
In this study we asked the question of whether a specific interaction can be based primarily on structural recognition. The trp repressor/operator complex as revealed from the crystallographic structure has only a few direct and indirect hydrogen bonds. On the other hand it contains many hydrogen bonds to the sugar-phosphate backbone of the DNA, which is thought to reflect the three-dimensional structure of the DNA sequence. Another aspect of this interaction is the significant distortions of the operator from standard B-DNA structure. Therefore, the trp repressor/operator complex is a good candidate for a model system for specific interactions based primarily on the structural parameters of the operator sequence. To test this hypothesis we selected the DNA sequences that bind the trp repressor from a pool of random sequences, by separating the different complexes with the trp repressor from one another, and from the free DNA, by gel electrophoresis.
From the analysis of the motifs characterizing the selected sequences we can conclude that the basis for specific interaction between the trp repressor with its target site, is direct and indirect readouts, whereas structural recognition has mainly an assisting role.