Team:ETH Zurich/UVR8

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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 <span class='eth_reference'>[Heijde 2012]</span>. At the dimeric interface UVR8 contains tryptophane residues which act as chromophore absorbing UV-B light and absorbed energy is used to break cross-dimer salt bridges resulting in the dissociation of the dimer <span class='eth_reference'>[Christie 2012]</span>. The monomeric UVR8 is then able to initiate downstream reactions that trigger the transcription of proteins needed for the stress response <span class='eth_reference'>[Heijde 2012]</span>.  
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 <span class='eth_reference'>[Heijde 2012]</span>. At the dimeric interface UVR8 contains tryptophane residues which act as chromophore absorbing UV-B light and absorbed energy is used to break cross-dimer salt bridges resulting in the dissociation of the dimer <span class='eth_reference'>[Christie 2012]</span>. The monomeric UVR8 is then able to initiate downstream reactions that trigger the transcription of proteins needed for the stress response <span class='eth_reference'>[Heijde 2012]</span>.  
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We want to make use of UVR8 and incorporate it into our system in ''E.coli'' to create a novel UV-B responsive transcription factor. Since UVR8 is not able to interact with DNA itself <span class='eth_reference'>[Cloix 2008]</span>, we fused it with the DNA binding domain (DBD) of a well characterized tetracycline repressor protein TetR. Naturaly, TetR forms a homodimer which binds and repress TetR responsive promoter P<sub>tet</sub>. However,sudgested TetR<sub>DBD</sub> lacks of dimerization domain and residual monomer unable to bind its promoter <span class='eth_reference'>[Thibodeaux 2009]</span>. Thus, TetR<sub>DBD</sub>-UVR8 chimera behaves in two ways: UVR8 provides UV-B susceptible dimerization, while TetRDBD lets fusion protein to repress P<sub>tet</sub> . Upon UV-B irradiation, UVR8 dimer is broken and TetR<sub>DBD</sub>-UVR8 is released from P<sub>tet</sub>  activating transcription, thus creating UV-B-ON switch in ''E.coli''.   
+
We want to make use of UVR8 and incorporate it into our system in ''E.coli'' to create a novel UV-B responsive transcription factor. Since UVR8 is not able to interact with DNA itself <span class='eth_reference'>[Cloix 2008]</span>, we fused it with the DNA binding domain (DBD) of a well characterized tetracycline repressor protein TetR. Naturaly, TetR forms a homodimer which binds and repress TetR responsive promoter P<sub>tet</sub>. However,sudgested TetR<sub>DBD</sub> lacks of dimerization domain and residual monomer unable to bind its promoter <span class='eth_reference'>[Thibodeaux 2009]</span>. Thus, TetR<sub>DBD</sub>-UVR8 chimera behaves in two ways: UVR8 provides UV-B susceptible dimerization, while TetR<sub>DBD</sub> lets fusion protein to repress P<sub>tet</sub> . Upon UV-B irradiation, UVR8 dimer is broken and TetR<sub>DBD</sub>-UVR8 is released from P<sub>tet</sub>  activating transcription, thus creating UV-B-ON switch in ''E.coli''.   

Revision as of 21:17, 26 September 2012

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Navigate through Overview, Design and Results


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 [Heijde 2012]. At the dimeric interface UVR8 contains tryptophane residues which act as chromophore absorbing UV-B light and absorbed energy is used to break cross-dimer salt bridges resulting in the dissociation of the dimer [Christie 2012]. The monomeric UVR8 is then able to initiate downstream reactions that trigger the transcription of proteins needed for the stress response [Heijde 2012].

We want to make use of UVR8 and incorporate it into our system in E.coli to create a novel UV-B responsive transcription factor. Since UVR8 is not able to interact with DNA itself [Cloix 2008], we fused it with the DNA binding domain (DBD) of a well characterized tetracycline repressor protein TetR. Naturaly, TetR forms a homodimer which binds and repress TetR responsive promoter Ptet. However,sudgested TetRDBD lacks of dimerization domain and residual monomer unable to bind its promoter [Thibodeaux 2009]. Thus, TetRDBD-UVR8 chimera behaves in two ways: UVR8 provides UV-B susceptible dimerization, while TetRDBD lets fusion protein to repress Ptet . Upon UV-B irradiation, UVR8 dimer is broken and TetRDBD-UVR8 is released from Ptet activating transcription, thus creating UV-B-ON switch in E.coli.



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.

References

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