Team:ETH Zurich/UVR8

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UVR8

Figure 1: UVR8 as a symmetric homodimer. Upon UV-B exposure the the dimer dissociates into two monomers.

UVR8 is a plant protein first identified in Arabidopsis thaliana.  UVR8 is a photoreceptor and a crucial part in the plant stress response to UV-B (280-315 nm).  In absence of UV-B the protein occurs as a dimer. At the dimeric interface it contains tryptophane residues which absorb the UV-B light and thereby changing conformation of the protein  which results in the dissociation of the dimer. The monomeric UVR8 is then able to initiate downstream reactions that trigger the transcription of proteins needed for the stress response.

We want to make use of this photoreceptor by incorporating it into our system in E.coli as a transcription factor. Since UVR8 is not able to interact with DNA by itself we are fusing it to the DNA binding domain (DBD) of a TetR monomer. TetR is a well characterized repressor. We make use of the fact that the Tet repressor is unable to bind the DNA as a monomer. UVR8 fused to the TetR DBD hence behaves as a TetR homodimer and is thus able to repress the ptet promoter in the dark state. As the UVR8 dimer is broken upon UV radiation, also the TetR DBD's dissociate from each other and cannot occupy the ptet promoter any longer. Hence transcription of the genes downstream to that promoter can start.

dTetR a novel two-hybrid screening in E.coli

Figure 2: Novel two-hybrid screening: TetR DNA binding domain fused with the target proteins A,B or C.

TetR is a transcriptional regulator, controlling the expression of tetracycline resistance in E.coli. TetR binds as a homodimer to the operator and acts as a repressor. Each monomer consists of a DNA binding domain at the N-terminal end as well as a core domain, important for the dimerization, and a ligand binding domain.

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