TraR bound to autoinducer and target DNA</scene>' scene='Insert optional scene name here' />
Description
Shown is bound to its autoinducer (OOHL) [1] and
TraR is a quorum sensing protein in Agrobacterium tumefaciens. Shown is TraR bound to its autoinducer 3-oxooctanoyl-homoserine lactone (OOHL) [1], also called Agrobacterium autoinducer (AAI) [2], and its target DNA.
Quorum sensing is used by bacteria to regulate gene expression depending on cell-population density [3]. Therefore, Bacteria use small hormone-like proteins called autoinducers [4]. These autoinducers increase in concentration in connection to increasing cell density [3]. Reaching a minimal threshold stimulatory concentration, the autoinducers activate gene regulation processes [3]. This quorum sensing becomes beneficial as soon as it is performed by many cells [4]. Quorum sensing is used by Gram-negative as well as Gram-positive bacteria and occurs within and between bacterial species [3]. The communication via quorum sensing may have been a first step of multi-cellularity and makes the distinction between eukaryotes and prokaryotes more complex [3] [4].
TraR is member of the quorum-sensing transcription factor family called LuxR including the Helix-Turn-Helix motif [5], that is present in many DNA binding proteins, e.g. Cro, CAP or the λ-repressor [6]. In presence of its autoinducer AAI, TraR regulates genes connected to the tumor inducing (Ti) plasmid [1]. When a certain cell density of Agrobacterium tumefaciens is reached, the transfer of the Ti-plasmid is induced. [5] There are two proteins that influence this transfer: TraR and TraI. The TraI gene encodes AAI. The absence of AAI causes rapid proteolysis of TraR [1] , which implies, that AAI protects TraR from degradation [2]. TraR itself activates the Ti-plasmid tra genes [7].
Structure
General Structure
In general TraR works as a , but the monomers are differently elongated which leads to a general asymmetric structure of the dimer. Each TraR monomer consists of 234 amino acids. In this , binding the tra box are shown. A and C are in the same way elongated, as well as B and D. Each monomer possesses its own as well as a . Thus, all in all four autoinducer molecules are bound to the TraR proteins at one tra box.
The dimers AB and CD interact with each other via molecular interactions between B and C. Additionally, the palindromic sequence of the tra box causes a base stacking between the beginning and the end of the sequence.
As A and B are slightly different, there is an dimeric asymmetry that has two consequences for the function. At first, the of the two monomers have different positions. The N-terminal part of A is between the ligand-binding domain and the DNA-binding domain in the center of the protein. In opposition to that, the N-terminal part of B is located externally. Moreover, there is a at the surface of the dimer. Because of that, the DNA-binding domain forms a long and basic region for the interaction with DNA, whereas the C-terminal residues form a positively charged patch exposed to the solvent. This region might be involved in protein-protein interaction with TraM. [4?5?] TraM binds to TraR and prevents it from binding to the DNA and therefore prevents the activation of the transcription of the tra genes. [5]
Ligand binding domain
The includes the residues 1 to 162. The ligand AAI is surrounded by three α-helices (α3, α4 and α5) and a five-stranded antiparallel β-sheet. The order of this β-sheet is 2-1-5-4-3.
The involved residues for the ligand binding and therefore the with AAI are L40, Y53, W57, Y61, F62, D70, V73, W85, F101 and Y102.
On the other side of the β-sheet there are three more α-helices: α1 (residues 3 to 12), α2 (residues 17 to 32) and α6 (residues 145 to 162). The long α6-helix enables hydrophobic interactions with the corresponding α-helix on the other monomer. Next to this, the dimeric structure is also stabilized by interactions between the last part of α1 and the connecting turn between α4 and α5.
Following the ligand binding domain, the residues 163 to 175 form the to the DNA binding domain. The residues 166 to 169 are disordered and cannot be seen in the structural model.
DNA binding domain
The DNA binding domain contains four (α7, α8, α9, α10) and is provided by the C-terminal part of the protein by the residues 176 to 234. In monomer A or C, the first residues of α7, the last residues of α8 and the residues 232 to 234 form polar bonds with the N-terminal part of the ligand binding domain. In the B or D monomer, no such interactions were observed. Besides, helices α8 and α9 form the (Helix-Turn-Helix) motif.
Biological and Biotechnological Relevance
TraR is the activator for the conjugal transfer of Ti-plamids. [8] In nature, Agrobacterium tumefaciens uses the transfer of the Ti-plasmid to manipulate dicotyledonous plants into producing metabolites as nutrients for the bacteria. [7] TraR activates the tra genes if AAI is present and also activates the transcription of traR and traI. [8] The tra genes are responsible for the conjugal transfer.
In biotechnology, the whole transfer system of the Ti-plasmid can be used to transfer DNA into dicotelydonous plant cells and to integrate that DNA into the plant genome. Naturally, Agrobacterium tumefaciens only infects dicotelydonous plants and the systems to perform this are well established. But there were also some experiments that proved a transformation using the ti-plasmid in monocotyledons. [8]
Ligand:
Ligand Binding Site:
neg charge dna binding
References
- ↑ 1.0 1.1 1.2 1.3 Chai Y, Winsans SC (2005): Amino-terminal protein fusions to the TraR quorum-sensing transcription factor enhance protein stability and autoinducer-independent activity. In: J. Bacteriol. 187(4); 1219-26;
- ↑ 2.0 2.1 Vannini A. et al. (2002): The crystal structure of the quorum sensing protein TraR bound to its autoinducer and target DNA. In: EMBO Journal; Vol. 21; 4393-4401
- ↑ 3.0 3.1 3.2 3.3 3.4 Miller M.B., Bassler C. L. (2001): Quorum Sensing in Bacteria. In: Annu. Rev. Microbiol.; 55; 165-199
- ↑ 4.0 4.1 4.2 Waters C. M., Bassler C. L. (2005): Quorum Sensing: Cell-to-Cell Communication in Bacteria. In: Annu. Rev. Cell Biol.; 21; 319-346
