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. [Heijde2012]

UVR8 is a plant protein first identified in Arabidopsis thaliana. It 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 [Heijde2012]. UVR8 contains tryptophane residues forming a chromophore. In the dark state they interact with each other and thus UVR8 dimerizes. UV-B radiation interrupts this interaction and the UVR8 dimer falls apart [Christie2012]. Downstream reactions triggering transcription of essential proteins for the stress response cannot occur anymore [Heijde2012].

We will make use of that phenomenon by incorporating UVR8 into our system in E.coli as a UV-B responsive transcription factor. Therefore we fused UVR8 to the TetRDBD, the TetR DNA binding domain. The TetRDBD can bind to the Ptet promoter as a dimer only. Thus the UVR8 TetRDBD fusion behaves like a TetR homodimer, able to repress the Ptet promoter in the dark state. As the UVR8 dimer is broken upon UV-B radiation, the TetRDBD occurs as an inactive monomer and cannot occupy the Ptet promoter any longer. Hence transcription of the genes controled by the Ptet promoter can start.



TetRDBD - 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.

To our prediction, dimerisation is needed for TetR efficient DNA binding due to cooperativity, thus the fusion of monomeric truncated TetR (TetRDBD) version with an extraneous dimerization domain, should restore TetRDBD DNA binding. This can easily be used as a two hybrid system to detect homo/heterodimers in E. coli, and, in principle, one can use these fusions to turn protein-protein interaction into the repression of transcription. To do this one can fuse the target proteins with the TetRDBD and if the proteins (A,C) can interact the output is repressed (see figure 2). Furthermore, inducible dimerization can lead to a novel switch like behavior of the system.

You can find the split [http://partsregistry.org/Part:BBa_K909007 TetRDBD ] containing a BamHI restriction site which can be fused to any protein of interest containing the corresponding restriction site in the DNA sequence. This allows you to test if a protein of interest is able to dimerize.

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