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Team:TUDelft/Temperature design2
From 2008.igem.org
>> Work in progress
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Changing the temperature threshold of the RNA thermometer
The prinriple of the RNA thermometer is based on base pairing between the nucleotides in the Shine Dalgarno region. A rise in temperature weakens the binding forces and above a threshold temperature the base-pairing forces are to low to hold the two nucleotides together.
In order to be able to design an RNA thermometer with a specific temperature threshold, we need to know which factors are of influence on this threshold. The most important one is probably the secondary structure around the Shine Dalgarno sequence. The basic principle of an RNA thermometer is that heat, or a rise in temperature, is needed to expose the Shine Dalgarno sequence by melting the part of the secondary structure containing this sequence. It sounds logical that the temperature threshold can be increased by incorporating nucleotides that form strong base pairs within the Shine Dalgarno region. This will result in a more stable helix for which a higher temperature is needed to make it unstable, i.e. melt. Incorporating nucleotides that has weaker base-pairing strength within the Shine Dalgarno region will have the opposite effect. The helix will become less stable and less heat will be needed to melt it. This way the temperature threshold at which the translation will initiate will drop.
Two facts that enforce this assumption... First, aligning the sequences of the different types of RNA thermometers produces conserved regions around the SD region. Second, it is shown by De Smit and Van Duin (reference) that there is a strong correlation between translation efficiency and the stability of the helix containing the Shine-Dalgarno region and the initiation codon.
But will this region on itself be enough to have a working RNA thermometer? Looking at the consensus structure of the ROSE and PrfA RNA thermometer, it can be seen that there are other regions within the structure that are highly conserved. This indicates that these regions might also be of importance to the functioning of the RNA thermometer. It is still unknown if the stem-loops not containing the SD are of any help with the opening of the SD region (reference).
Pseudoknot analysis.
figures...(consensus structures, highly conserved SD regions)
De Smit and Van Duin [1] show a correlation between the level of expression and the free energy of the secondary structure in the Shine Dalgarno region.
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References
- ^ De Smit M H, Van Duijn J (1990). "Secondary structure of the ribosome binding site determines translation efficiency: A quantitative analysis". PNAS, 1990-10, vol.87, no.19, 7668-7672. PMID:2217199
- ^ Hoe N P, Goguen J D (1993). "Temperature sensing in Yersinia pestis: Translation of the LcrF activator protein is thermally regulated". J Bacteriol, 1993 December, 175(24), 7901-7909. PMID:7504666
- ^ 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. PMID:16710302
- ^ Nocker A, Hausherr T, Balsiger S, Krstulovic N, Hennecke H, Narberhaus F (2001). "A mRNA-based thermosensor controls expression of rhizobial heat shock genes". Nucleic Acids Research, 2001 December 1, 29(23):4800-4807. PMID:11726689
- ^ Balsiger S, Ragaz C, Baron C, Narberhaus F. "Replicon-specific regulation of small heat shock genes in Agrobacterium tumefaciens". Journal of Bacteriology, October 2004, p. 6824-6829, Vol.186, No.20. PMID:15466035
- ^ 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. PMID:17630972
- ^ Johansson J, Mandin P, Renzoni A, Chiaruttinni C, Springer M, Cossart P. "An RNA thermosensor controls expression of virulance genes in Listeria monocytogenes". Cell , Volume 110 , Issue 5 , 551-561. PMID:12230973
