Team:ETH Zurich/UVR8

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==UVR8==  
==UVR8==  
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[[Image:UVR8.png|frameless|220px|right|thumb|Figure 1: UVR8 as a symmetric homodimer. Upon UV-B exposure the the dimer dissociates into two monomers.
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[[Image:UVR8_2.png|frameless|350px|right|thumb|Figure 1: UVR8 as a symmetric homodimer. Upon UV-B exposure the the dimer dissociates into two monomers.
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<span class='eth_reference'>[Heijde2012]</span>]]
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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). 
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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.
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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 <sub>DBD</sub> hence behaves as a TetR homodimer and is thus able to repress the P<sub>Tet </sub> promoter in the dark state.
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As the UVR8 dimer is broken upon UV radiation, also the TetR<sub>DBD</sub>'s  dissociate from each other and cannot occupy the P<sub>Tet </sub>promoter any longer. Hence transcription of the genes downstream to that promoter can start.
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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 <span class='eth_reference'>[Heijde2012]</span>. 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 <span class='eth_reference'>[Christie2012]</span>. Downstream reactions  triggering transcription of essential proteins for the stress response cannot occur anymore <span class='eth_reference'>[Heijde2012]</span>.
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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 TetR<sub>DBD</sub>, the TetR DNA binding domain. The TetR<sub>DBD</sub> can bind to the P<sub>tet</sub> promoter as a dimer only. Thus the UVR8 TetR<sub>DBD</sub> fusion behaves like a TetR homodimer, able to repress the P<sub>tet</sub> promoter in the dark state. As the UVR8 dimer is broken upon UV-B radiation, the TetR<sub>DBD</sub> occurs as an inactive monomer and cannot occupy the P<sub>tet</sub> promoter any longer. Hence transcription of the genes controled by the P<sub>tet</sub> promoter can start.
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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.  
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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To our prediction, dimerisation is needed for TetR efficient DNA binding due to cooperativity, thus the fusion of monomeric truncated TetR (TetR-DBD) version with an extraneous dimerization domain, should restore tetR-DBD DNA binding.   
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To our prediction, dimerisation is needed for TetR efficient DNA binding due to cooperativity, thus the fusion of monomeric truncated TetR (TetR<sub>DBD</sub>) version with an extraneous dimerization domain, should restore TetR<sub>DBD</sub> DNA binding.   
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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 TetR<sub>DBD</sub> 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.  
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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 TetR<sub>DBD</sub> 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.  
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Constructs were tested in two plasmid system: repressor and reporter plasmids.
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As repressor plasmid, a medium copy number pSEVA183 derived plasmid (provided by Andreas Bosshart), containing constitutively expressed LacI, were used. TetR<sub>DBD</sub>-UVR8 fusions and TetR<sub>DBD</sub> and full length TetR for control measurements were cloned after LacI a regulating P<sub>tac</sub> promoter.
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For the reporter plasmid, the GFP producing biobrick from P<sub>tet</sub> promoter (<partinfo>BBa_I13522</partinfo>) was cloned into low copy number pSB4K5 plasmid. 
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You can find the split [http://partsregistry.org/Part:BBa_K909007 TetR<sub>DBD</sub>  ] 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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Latest revision as of 19:43, 26 October 2012

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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.

References

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