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content.text= "<b> Introduction: </b> </br>The ClpXP protein complex is an AAA+ protease, which means that it uses the energy of ATP hydrolysis to unfold and degrade marked proteins. The genetic code of this complex is highly conserved and can be found in human cells as well as in the bacteria Escherichia coli. The degradation system was discovered in the early 1990's and is now well established ^{<a href=#131>13.1</a>}.In our project we used ClpXP to degrade specific proteins in order to control their amount and effect. Therefor we utilized the common adaptor sspB. This protein recognizes substrates tagged with ssrA . In order to achieve a better control we made use of a sspB split system. For more detailed information about the ClpXP degradation system in our project go to ClpXP general. </br> </br> <b> Structure:  </b> </br> The ClpXP complex consists of two functional and structural different parts. The ClpX protein, an ATPase, is a hexameric ring (Fig.1) with a pore in the center<div align='lef'><img src='https://static.igem.org/mediawiki/2013/9/98/Bonn_Clp_Fig1.jpg' height='348' width='320'>Fig. 1: the hexameric ring of ClpX, each color represents a subunit, from  &quot;ClpXP, an ATP-powered unfolding and protein-degradation machine, Baker et al, Biochim Biophys Acta, 2012, PMCID: PMC3209554&quot;</div> </br> Each subunit contains a N-terminal domain (Fig.2, B), which assumes the adaptor recognition and is stabilized by coordinated zinc atoms.However, the important part of a subunit is the AAA+ module (Fig.2, C), divided in a large and a small domain. <div align='left'><img src='https://static.igem.org/mediawiki/2013/d/d0/BonnClp_Fig2.jpg' height='262' width='499'>Fig. 2: structure of a ClpX subunit, B: the N-terminal domain with brown zinc atoms, C: the AAA+ module, from  &quot;ClpXP, an ATP-powered unfolding and protein-degradation machine, Baker et al, Biochim Biophy Acta, 2012, PMCID: PMC3209554&quot;</div> </br> Between these domains the ATP binding site can be found, but not every subunit is able to bind the nucleotide. The arrangement of ATP binding and not-binding subunits in the hexameric ring is essential for the tertiary structure and the conformation changes after hydrolysis. The ClpP protein is a tetradecameric peptidase (Fig. 3, A and B).The subunits are arranged as two heptameric rings, one ring stacked on top of the other, with also a narrow pore in the center. This pore leads into the proteolytic chamber, which is barrel-shaped. Every subunit accommodates a classical Ser-His-Asp catalytic triad and oxyanion hole inside the chamber. Those proteolytic acitve sites (Fig. 3, C) can form several hydrogen bonds to the substrate ^{<a href=#131>13.1</a>}. <div align='left'><img src='https://static.igem.org/mediawiki/2013/0/06/BonnClp_Fig3.jpg' height='401' width='382'>Fig. 3: structure of ClpP, A: side view with stabilizing residues (blue), B: top view with the pore (red), C: active site of a subunit with a bonded substrate, from &quot;ClpXP, an ATP-powered unfolding and protein-degradation machine, Bakeret al, Biochim Bi phys Acta, 2012, PMCID: PMC3209554&quot;</div> </br> </br> </br><b> Functions: </b> </br>The ClpXP complex has three tasks to fulfill: </br> </br>1. Binding: The substrate binding process at the ClpX unit is normally conducted with the aid of an adaptor protein.This protein identifies tagged substrates and delivers them to the complex (Fig. 4, left). In order to transfer the protein, the adaptor also binds to the ClpX unit (Fig. 4, right), so that parts of the tag get approximated to a special binding site on the complex. After the linking between the tag and the binding site has been performed, the unfolding starts.The binding process also works without an adaptor protein, but an adaptor enhances the degratation by improving enzyme-substrate affinity. </br> </br> 2. Unfolding and translocation: The translocation of polypeptids through the ClpX unit to the ClpP chamber is an active process using energy from ATP-binding and -hydrolysing cycles. Therefor are several ATP molecules linked to the ClpXprotein. The separation of one phosphate molecule results in conformation changes, which pulls the linked protein more inside the pore located in the center of ClpX. The remaining ADP has to be replaced by a new ATP molecule before the cycle can start again. Meanwhile the unfolding is driven automatically, because the large tagged protein has to fit into the narrow pore, which forces the three-dimensional structure to become linear. </br> </br> 3. Degradation: The axial pore of the ClpP unit is also very narrow, allowing the entry of only small unfolded peptides into the proteolytic chamber. Inside the chamber, the substrate binds to an active site over several hydrogen bonds. It also can be linked to multiple active sites. In this position, proteins are cleaved in a maximum speed of around 10,000 proteins per minute by ClpP alone. If the ClpX unit is added, the rate is with ~0.2 proteins per minute and 0.3 &my;M substrate much lower, because the unfolding process takes more time ^{<a href=#131>13.1</a>} ^{<a href=132>13.2</a>}. </br> </br> <div align='left'><img src='https://static.igem.org/mediawiki/2013/b/b8/BonnClp_Fig4.jpg' height='311' width='628'>Fig. 4: Model of the degradation process with the sspB adaptor, from &quot;Engineering controllable protein degradation, McGinness et al, Molecular cell, 2006, PMID:16762842'</div> <h2><b> References </b> </h2></br> </br> <p><a name=131>13.1</a> ClpXP, an ATP-powered unfolding and protein-degradation machine, Baker et al, Biochim Biophys Acta, 2012, PMCID: PMC3209554</br> <a name=132>13.2</a> Flexible Linkers Leash the Substrate Binding Domain of SspB to a Peptide Module that Stabilizes DeliveryComplexes with the AAA ClpXP Protease, Wah et al, 2003, Molecular cell, PMID: 14536075</p></br>";  

content.text= "The family of the &quot;zinc finger&quot; proteins has a different approach of Transcription regulation. They contain a zinc ion as a cofactor. Zinc finger proteins have a DNA binding and a dimerization domain. They can be differentiated due to different loops. On the one hand they can bind to almost every part of DNA and on the other hand they can bind to several receptors. Hence the activating or repressing effect is not defined by the zinc finger itself, but by the effector Protein it is binding to^{<a href=#721>72.1</a>}. </br></br> <div class='content-image'> <img src='https://static.igem.org/mediawiki/2013/4/4b/BonnZincFinger.jpg' width='400' height='400'></br>&quot;The zinc ion (green) is coordinated by two histidine and two cysteine amino acid residues&quot;^{<a href=#722>72.2</a>}</div></br></br> <p><a name=721>72.1</a> <a href=http://www.pnas.org/content/early/2013/09/11/1303625110.long> Transcription factor ZBED6 affects gene expression, proliferation, and cell death in pancreatic beta cellsXuan Wang, Lin Jiang,Ola Wallerman, Ulla Engström, Adam Ameur, Rajesh Kumar, Gupt, YuQi, Leif Andersson and Nils Welsh Science for Life Laboratory, Department of Medical Cell Biology, and Science for Life Laboratory, Department of Medical Biochemistry and Microbiology, Uppsala University, SE-75123 Uppsala, Sweden; Ludwig Institute for Cancer Research Ltd., Science for Life Laboratory, Uppsala University, SE-751 24 Uppsala, Sweden; andScience for Life Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, SE-75185 Uppsala, Sweden </a> </p> </br> <p><a name=722>72.2</a> <a href='http://en.wikipedia.org/wiki/Zinc_finger'>Cartoon representation of the zinc-finger motif of proteins</a></p>";

content.text= "Tales are proteins which bind promoter sequences. Their DNA binding domain consists of several tandem repeats that are able to bind specific domains of the DNA. These tandem repeats can be engineered easily. Thus the user can define the domain to bind to. Very similar to the zinc finger they actually do not regulate transcription but bind effector proteins which are able to activate transcription. The great advance, in comparison to the zinc finger domain, is it's easy way of engineering. Scientists can regulate very specificlly transcription by the use of TALEs.</br></br><p> <a href='http://onlinelibrary.wiley.com/doi/10.1111/jipb.12091/abstract'> Site-Specific Gene Targeting Using Transcription Activator-Like Effector (TALE)-Based Nuclease in Brassica oleracea: Zijian Sun†,Nianzu Li†, Guodong Huang, Junqiang Xu, Yu Pan, Zhimin Wang, Qinglin Tang, Ming Song*, Xiaojia Wang> </a> </p>";

content.text= "SsrA and sspB are peptides that mediate proteolysis via the ClpXP protease system in bacteria. In this article and the related articles, the focus is on their orthologs in C. crescentus, being referred to as ^CcssrA and ^CcsspB&alpha;, respectively, omitting ^Cc when obvious out of context. The ClpXP protease has an important function in regulation of the cell division cycle by effective proteolysis of short-lived regulatory proteins.</br>A protein which needs to be degraded will be tagged with the amino acid peptide ^CcssrA which is added at its C-terminus during translation. ^{<a href=#741>74.1</a>, <a href=#742>74.2</a>} The ClpX subunit of the ClpXP protease recognizes the ssrA tag by specific binding and unfolds the tagged protein, in which ATP is hydrolyzed. In C. crescentus, the ssrA tag has a length of 14 amino acids, while the E. coli ortholog is only eleven amino acids long. </br>sspB&alpha; is a dimeric protein that serves as a tether which brings the ssrA tagged protein and the ClpXP protease together and therefore accelerates protein degradation. It simultaneously binds to both the ssrA tag and the ClpX subunit and in this way brings the tagged protein in close contact with the protease. </br></br><h2>References</h2></br><a name=741>74.1</a> <a href='http://www.ncbi.nlm.nih.gov/pubmed/11535833'>Overlapping recognition determinants within the ssrA degradation tag allow modulation of proteolysis, Flynn et al., Proceedings of the National Academy of Sciences of the United States of America, 2001, PMID: 11535833</a></br><a name=742>74.2</a> <a href='http://www.ncbi.nlm.nih.gov/pubmed/17937918'> Structure and substrate specificity of an SspB ortholog: design implications for AAA+ adaptors, Chien et al., Cell Press, 2007, PMID: 17937918</a>";