"
Team:TUDelft/Temperature analysis
From 2008.igem.org
>> work in progress
Contents |
Analysis
Introduction
Cells are constantly subjected to changing environmental conditions and one example of such a changing environmental condition is temperature. A mechanism found in different organisms, that makes the cell respond to thermal changes, is the RNA thermometer. For the input of our system we are going to use this mechanism to let the cell produce a color or a smell at a certain temperature.
Background
RNA switch
A way to respond to environmental changes is through [http://en.wikipedia.org/wiki/Transcriptional_regulation transcriptional regulation]. This, most well known regulation system, acts at the DNA level in which proteins or protein complexes regulate the transcription of certain genes by binding to the DNA.
Recently a number of regulatory systems that work at the RNA level has been discovered. These systems, which are called RNA switches, regulate the translation instead of the transcription. They all work in a similar way. In a certain state the RNA is folded in such a way that the Shine Dalgarno region (ribosome binding site) is occluded, preventing the ribosome to bind to the RNA and thereby preventing the initiation of the translation. In this case you could say that the switch is in the off-state, which means that the translation of the gene encoded by the RNA stretch is switched off.
An external factor can cause a state transition from the off to the on state. This happens through a conformational change of the RNA caused by the external factor. After the conformational change the Shine Dalgarno is exposed, enabling the ribosome to bind to the RNA and thereby enabling the translation of the protein encoded by the RNA.
RNA thermometer
There are different RNA switches having different factors that 'switch the system on'. For example, RNA switches that are switched on by small ligands are called riboswitches and those that are switched on by short trans-RNA stretches are called trans-acting RNA switches. The ones we are interested in are the RNA thermometers. These RNA switches respond to a change in temperature. When the temperature rises above a certain threshold, the hairpin region around the Shine Dalgarno will melt and become exposed. This way a rise in temperature can cause the initiation of translation.
RNA thermometers reside at the 5' end of an mRNA of a protein. This 5' non-coding mRNA region forms a structure that blocks the translation by occluding the Shine-Dalgarno region at a certain temperature, e.g. 30 degrees Celcius. When the temperature rises above the threshold temperature, e.g. 37 degrees, a conformational change of the structure (melting of part of the hairpin surrounding the Shine-Dalgarno region) will cause the Shine-Dalgarno to become exposed, enabling the ribosome to bind to the mRNA and initiate the translation of the the protein encoded by the mRNA (figure x).
RNA thermometer families
When we look at known RNA thermometers (the research area is relatively young and it is expected that more are to be found) they can be split up into different families based on their [http://en.wikipedia.org/wiki/RNA_structure secondary structure]. Two of these families: Rose and PrfA, as specified by the [http://rfam.sanger.ac.uk/ Rfam database], are found in procaryotes and thus of interest to us. A third family is found in literature and is proposed to be called the FourU family [number] .
References
- ^ Waldminghaus T, Heidrich N, Branti S, Narberhaus F (2007). "FourU: a novel type of RNA thermometer in Salmonella". Molecular Microbiology, Volume 65, Issue 2, 413-424. [http://www.ncbi.nlm.nih.gov/pubmed/17630972 PMID:17630972]
Phase I: Introducing an RNA thermometer into e. Coli
Intro...
Three existing RNA thermometers
As a first approach we will turn three existing RNA thermometers into a biobrick in order to test if we can incorporate an artificial temperature sensitive system into e. coli. We selected three different RNA thermometers from literature which have been proven to be working RNA thermometers.
ROSE
Repressor Of heat-Shock gene Expression (ROSE) is the (conserved) mRNA sequence found in front of some prokaryotic heat-shock proteins [number]. Turning this into a biobrick should allow induction of translation by heating to 42 °C.
The first RNA thermometer is one of the ROSE family and is retrieved from the organism Bradyrhizobium japonicum. ... The second RNA thermometer is part of the FourU family... retrieved from Salmonella... The third RNA thermometer is retrieved from the Listeria monocytogenes and belongs to the PrfA family. The switching temperature is at 37 degrees Celsius.
Virulence derived expression
Various pathogenic microorganisms express virulence proteins only inside a host. It has been shown in the 90's this is induced by the increased temperature having effect on translation but not on transcription [http://jb.asm.org/cgi/reprint/175/24/7901]. Examples of microorganisms using this temperature induced virulence are Salmonella [http://www3.interscience.wiley.com/cgi-bin/fulltext/118542064/PDFSTART], Yersinia pestis or Listeria monocytogenes. Of course we won't work with these virulent genes, only with the regulating mRNA sequences in front of them or their TF. An induction temperature of 37 °C seems logical.
(Designed)G-Quadruplex structures
As described by Wieland et al. [http://www.sciencedirect.com/science?_ob=MImg&_imagekey=B6VRP-4P8YWVM-7-8&_cdi=6240&_user=499885&_orig=search&_coverDate=07%2F30%2F2007&_sk=999859992&view=c&wchp=dGLbVzz-zSkzS&md5=97741390ae4ff0d9a8c4d5f41beb0f1d&ie=/sdarticle.pdf] it is possible to design sequences that form tertiary structures occluding the RBS. A temperature range of 30 - 35 °C has been achieved.
Artificial RNA thermometer based on G-quadruplex
A fourth working RNA thermometer found in literature is an artificial one. It is based on a special tertiary (3D) structure in which an RNA stretch can fold. This structure also occludes the Shine Dalgarno region and thereby blocks the translation. Above a certain threshold temperature the structure becomes unstable and the Shine-Dalgarno becomes exposed, enabling the ribosome to bind to the RNA and initiate the translation process. Although this artificial RNA thermometer is very interesting, it turned out to be impossible make a biobrick out of it. As can be read in the design part (link) the scar (the result of the ligation of the RNA thermometer part with the protein coding part) has to be introduced in the RNA thermometer. Unfortunately the introduction of the scar changes the structure that makes the part temperature sensitive. Therefor this G-quadruplex RNA thermometer is not taken into account any further. Still it remains very interesting, because it shows that an artificial thermometer can be made using only the structure of the RNA.
References
- ^ Chowdhurry S, Maris C, Allain F H T, Narberhaus F (2006). "Molecular basis for temperature sensing by an RNA thermometer". The EMBO Journal, 2006, 25, 2487–2497. [http://www.ncbi.nlm.nih.gov/pubmed/16710302 PMID:16710302]
Phase II: Changing the temperature threshold of the RNA thermometer
References
</div>
</div>
