CH391L/S13/Riboswitches

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DNT (2,4-dinitrotoulene) is used in the production of bedding and cushioning in furniture and is also an intermediate chemical in the production of TNT. DNT is hazardous and can contaminate bodies of water as well as soil. The detection of DNT is important to prevent contamination but also to detect unwarranted production of TNT.  
DNT (2,4-dinitrotoulene) is used in the production of bedding and cushioning in furniture and is also an intermediate chemical in the production of TNT. DNT is hazardous and can contaminate bodies of water as well as soil. The detection of DNT is important to prevent contamination but also to detect unwarranted production of TNT.  
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E. Coli can be used to detect DNT by fluorescence. A fluorescence resonance energy transfer (FRET) riboswitch was created by placing eFGP (enhanced green fluorescent protein) and a REACh (Resonance Energy Accepting Chromoprotein) in combination along the mRNA strand with a TEV protease linking two proteins. In the presence of DNT the riboswitch is activated and the TEV protease is translated. The protease cleaves the link between eFGP and REACh thus allowing eFGP to become fluorescent. In this way, DNT can be detected by the fluorescence of E. Coli.
E. Coli can be used to detect DNT by fluorescence. A fluorescence resonance energy transfer (FRET) riboswitch was created by placing eFGP (enhanced green fluorescent protein) and a REACh (Resonance Energy Accepting Chromoprotein) in combination along the mRNA strand with a TEV protease linking two proteins. In the presence of DNT the riboswitch is activated and the TEV protease is translated. The protease cleaves the link between eFGP and REACh thus allowing eFGP to become fluorescent. In this way, DNT can be detected by the fluorescence of E. Coli.

Revision as of 18:56, 24 February 2013

Riboswitches


Contents

What a riboswitch is and how it works


A riboswitch is a region on the mRNA that can act like a switch to turn gene expression on or off. In the presence of a specific ligand, a riboswitch changes shape uncoupling the polymerase of the DNA. Polymerase is no longer able to transcribe thus terminating gene expression. Riboswitches are useful for gene regulation.


The mRNA has the ability to fold and base pair with itself forming different structures, such as those found on the regions that make up a riboswitch. On a basic riboswitch, there are two main regions. One region is more sensitive to ligands, also known as an aptamer. The second region is also known as a terminator hairpin and its existence is dependent on whether or not a ligand is bound to the aptamer. When a ligand binds to an aptamer it causes the aptamer to become more stable. Because a more stable aptamer region forms, the mRNA downstream from the aptamer undergoes a shape change and a terminating hairpin forms via mRNA base pairing with itself. In the event that a ligand has bound to an aptamer and a hairpin has formed, transcription is terminated by the uncoupling of the DNA polymerase. mRNA is transcribed until the polymerase reaches a polyuracil region down stream of the riboswitch. The polyuracil region is also known as a pause site because the polymerase tends to slow down as it passes through. The polymerase is knocked of the DNA by the hairpin as it reaches the polyuracil region and uncouples thus terminating transcription. If a ligand does not bind to the aptamer region, the polymerase will simply continue transcribing down the DNA strand and through the pause site.

A little history

Riboswitches were first discovered around 2002. Metabolic pathways and such were noticed to change in the presence of certain chemistry such as vitamin B but it was not understood why this occurred and how it changed gene expression. It was discovered that vitamin B acts as a ligand and binds to the aptamer on the cobalamin riboswitch. With this discovery more naturally occurring riboswitches were discovered. The discovery of riboswitches allowed for a better understanding of how the DNA is able to regulate its own gene expression.

Types of riboswitches and surrounding conditions

Naturally occurring riboswitches usually terminate gene expression when bound by a ligand and have only two domains: an aptamer and a terminating hairpin. There are riboswitches that can turn gene expression on when bound by a ligand such.


Some riboswitches are not dependent on a ligand but rather temperature. An example is a magnesium transport protein riboswitch found in Salmonella enterica. When a certain temperature is reached, the cell experiences heat shock and transcription of the mgtA gene is turned on. Increased transport of magnesium is thought to have thermoprotective functions by diffusion of energy and oxygen thus protecting the cells membrane and proteins from denaturing.


Like other molecules, riboswitches are sensitive to pH levels, salt concentration, and temperature.

Research

DNT detector

DNT (2,4-dinitrotoulene) is used in the production of bedding and cushioning in furniture and is also an intermediate chemical in the production of TNT. DNT is hazardous and can contaminate bodies of water as well as soil. The detection of DNT is important to prevent contamination but also to detect unwarranted production of TNT.


E. Coli can be used to detect DNT by fluorescence. A fluorescence resonance energy transfer (FRET) riboswitch was created by placing eFGP (enhanced green fluorescent protein) and a REACh (Resonance Energy Accepting Chromoprotein) in combination along the mRNA strand with a TEV protease linking two proteins. In the presence of DNT the riboswitch is activated and the TEV protease is translated. The protease cleaves the link between eFGP and REACh thus allowing eFGP to become fluorescent. In this way, DNT can be detected by the fluorescence of E. Coli.

Theophylline

Theophylline is useful in medicine and has anti-inflammatory effects is used for respiratory diseases.

iGEM team ZJU from China used a theophylline riboswitch to test the efficiency of different tunable scaffolds that contained allosteric sites. The team named these alloscaffolds “clovers”. The more efficient the clover, the more fluorescent the E. Coli in the presence of the ligand theophylline.

The iGEM team at Imperial College at London discovered that an increase in theophylline transport rate and receptors increases the expression of the CheZ gene. CheZ has an affect on the way E. Coli swims thus by adjusting the theophylline levels the team was able to indirectly manipulate E. Coli’s swimming behavior.

Cocaine Detector

References

  1. , M., Findeiß, S., Weissheimer, N., Stadler, P. F., & Mörl, M. (2012). De novo design of a synthetic riboswitch that regulates transcription termination. Nucleic acids research, gks1330–. doi:10.1093/nar/gks1330 [Wachsmuth]
  2. , M. N., & Landry, D. W. (2002). Aptamer-Based Colorimetric Probe for Cocaine. Journal of the American Chemical Society, 124(33), 9678–9679. doi:10.1021/ja0259483 [Stojanovic]
  3. ’Connor, K., Fletcher, S. A., & Csonka, L. N. (2009). Increased expression of Mg(2+) transport proteins enhances the survival of Salmonella enterica at high temperature. Proceedings of the National Academy of Sciences of the United States of America, 106(41), 17522–7. doi:10.1073/pnas.0906160106 [O]
  4. , M., & Breaker, R. R. (2004). Gene regulation by riboswitches. Nature reviews. Molecular cell biology, 5(6), 451–63. doi:10.1038/nrm1403 [Mandal]
  5. , M. E., Harbaugh, S. V, Chushak, Y. G., Stone, M. O., & Kelley-Loughnane, N. (2013). Development of a 2,4-Dinitrotoluene-Responsive Synthetic Riboswitch in E. coli Cells. ACS chemical biology, 8(1), 234–41. doi:10.1021/cb300274g [Davidson]
  6. , P., Bernal-Perez, L. F., Annunziata, O., & Ryu, Y. (2012). Rationally-designed fluorescent lysine riboswitch probes. Organic & biomolecular chemistry, 10(39), 7872–4. doi:10.1039/c2ob26160j [Budhathoki]
  7. , K. F., & Breaker, R. R. (2006). Riboswitches as antibacterial drug targets. Nature biotechnology, 24(12), 1558–64. doi:10.1038/nbt1268 [Blount]
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