The blueprint for all life on Earth is encoded in two molecules, DNA (deoxyribonucleic acid) and RNA (ribonucleic acid). These molecules are organized into functional units known as genes. A collection of genes that comprises the blueprint for an organism is known as a genome.
The human genome is comprised of DNA like most other forms of life, from birds to bacteria. Viral genomes are more varied in their composition, and many viruses have RNA genomes. Genomes can be found in structural units called cells. The human body contains billions of cells, hence billions of copies of the same genome. By contrast, single-cell forms of life like Escherichia coli bacterium only contain one genome.
Think of DNA and RNA genomes as being enormous libraries that contain information on how each cell could work. Of course, to interpret these libraries, the cell needs a workforce to read it, understand it, and copy it. Complex molecules known as proteins play central roles in this work, copying the genome and decoding it into products required to support cell growth and thousands of other processes.
For most organisms, decoding DNA to make protein is a straightforward process: first, a protein called “RNA polymerase” reads a gene (a “book” from the library analogy), and builds an RNA from the instructions within. This RNA molecule is called a “messenger” and will then be read by another protein complex called the “ribosome”. The ribosome will “translate” the message that is contained in the messenger RNA and construct a protein from it. Thus, making any protein properly requires two faithful decoding steps (Figure 1).
Figure 1: Simplified view of how a bacterium (E. coli for instance) can produce proteins from its genome. An RNA polymerase “reads” a gene and produces a messenger RNA (mRNA) from it (1). The ribosome then decodes the mRNA and produces a protein (2).
You don’t walk into a library and read all books at once. Similarly, the cell does not decode all genes at the same time (or it would surely die!). Instead, many factors control the gene decoding process so that the cell will only make the right protein at the right time, and in the right amount. In fact, some proteins actually prevent gene decoding by attaching themselves to a gene and preventing the work of the RNA polymerase.
Other factors can also act on the messenger RNA to control gene expression. To be read by ribosomes, a messenger RNA needs to be folded in just the right way [1]. If you take a page from a book and crease it savagely, you will struggle to read it afterward. Similarly, ribosomes cannot decode messenger RNA if it is not folded properly. This folding is regulated by many factors; amongst them is the one the iGEM IONIS 2017 team has focused its attention on for its project “Softer Shock”: temperature [2]!
Temperature has an enormous influence on biological processes. Enzymes, proteins in charge of important processes in cells, typically perform best at a specific temperature, which impacts cell fate. For example, temperature-dependent enzymatic processes determine how big a cell will grow, and when a cell will divide. Temperature in the human body typically hovers around 37°C, so it should come as no surprise that our enzymes work best at this temperature. Likewise, the bacteria that live in our body as the gut bacteria Escherichia coli, also have enzymes that work best at 37°C. By contrast, other organisms that have evolved to grow in extremely hot environments can’t grow at lower temperatures. Therefore, if you put an organism out of its optimal temperature range, it will create stress and “shock” that might lead to death.
Most organisms have evolved survival mechanisms to help them endure the shock of extreme temperatures. The iGEM IONIS team was interested in a particular defense mechanism: a family of proteins called “cold-shock proteins” that help cells endure cold extremes. We focused on the model bacterium Escherichia coli to determine how it makes cold-shock proteins to survive cold temperatures. Indeed, at temperatures as low as 15°C, most cellular processes in Escherichia coli are severely slowed, but cold-shock proteins are still read from the bacteria’s ‘bookshelf’ [3].
According to various studies, the most important factor allowing these bacteria to decode specific cold-shock genes is…. RNA folding! The messenger RNA of cold-shock proteins does not fold properly at 37°C, so ribosomes cannot read it to make proteins. By contrast, at 15°C the messenger RNA folds properly, and can be decoded to make proteins [4]. This restricts cold-shock proteins to be made only in cells that are enduring low temperatures.
To understand what our team did, we need to explain the structure and function of messenger RNA further. A messenger RNA is composed of three core elements (let’s keep it simple!). The principal element is of course the “coding sequence” that is “translated” by the ribosome to give rise to a protein. Preceding this coding sequence is a very important element called “ribosome binding site” (RBS), which will permit the ribosome to attach itself correctly to the messenger RNA and start to assemble the protein. To access this ribosome binding site, the ribosome, much like a plane, needs a landing strip. This landing strip is found on the messenger RNA and is called the “untranslated region” (UTR) (Figure 2) [5].
Figure 2: Structure of a mRNA highlighting the role of each elements. The ribosome sub-unit 1 first “lands” on the UTR (1) and “search” for the RBS (2). When found, the sub-unit 1 recruits the sub-unit 2 (3) and the assembled ribosome “reads” the coding sequence (4), permitting the synthesis of a protein (5).
But why bother talking about all this? Patience now, we are getting to it! According to studies, it is the UTR of the cold-shock protein that makes the messenger RNA so unstable at high temperature and stable at low temperatures (Figure 3) [6]!
Figure 3: Simplified view of “cold-shock” protein synthesis at either low (1) or high (2) temperatures, dictated by the UTR folding. At 15°C, its particular “hairpin” shape facilitates the ribosome landing and therefore the protein synthesis. At 37°C, the absence of this pattern in the UTR shape prevents the ribosome landing and compromises the synthesis of any protein.
In a cell, every protein is encoded by a messenger RNA with unique UTR and RBS elements that enable protein synthesis. But what would happen if we replaced the UTR and RBS from a normal messenger RNA with elements from a ‘cold-shock’ protein-encoding mRNA? We could regulate any protein translation by the same mechanism: temperature!
This is exactly what we did in the laboratory for our project : we extracted the DNA sequence that encodes ‘cold shock’ UTR and RBS elements from the E.coli genome, and stitched it to the gene coding for AmilCP [7], a beautiful blue protein found in the coral species Acropora millepora. We hoped that our experiment would allow this protein to be made only in cold temperatures. The new hybrid gene was transferred back into E. coli in a “plasmid”, a circular mini-bookshelf (like a ‘ringlet’ of DNA) that is frequently used in molecular biology. Then the E. coli RNA polymerase could read it and make a brand new messenger RNA. Our hypothesis was that this messenger RNA, because it had this specific ‘cold-shock’ landing pad for ribosomes, would be unstable at high temperatures and would fail to make the blue AmilCP protein at 37°C. By contrast, at low temperatures the bacteria should turn blue (Figure 4) !
Figure 4: Global overview of the Softer Shock strategy to create a biological thermo-sensor.
In the experiment, we grew the E. coli containing our new engineered gene at 37°C, and then we split cells into two batches. One batch was put at 15°C and the other remained at 37°C. We waited 24h and collected them, and guess what? The E. coli batch at 15°C had turned blue and the other one had not (Figure 5)!
Figure 5: Expression of blue protein by E. coli bacteria added with our engineered gene at 37°C (a) and 15°C (b). This picture was taken in our lab during the iGEM competition.
Thus, we created a synthetic organism that could produce a blue reporter protein only in low temperatures. The blue protein itself is not that useful, but it provides an important ‘proof of concept’. In future studies, we could replace the blue protein with any other gene of interest. For example, we could engineer a bacterium to make a collection of proteins that could achieve a task at low temperatures, like detoxifying the environment or producing a biofuel. By contrast, the principles of our investigation could be used to regulate gene expression in the opposite way, creating genes that would only be expressed at higher temperatures (in such experiments these proteins will have to be tested to ensure that they can retain function at high temperatures as well). In the context of global warming, engineering bacteria that can make specific proteins in response to varied temperatures could have significant advantages. For example, such dual thermo-responsive gene expression systems could be used to provide protection to vegetables when applied on their surface. From a culture medium in a laboratory to your food stored in the freezer, think about the PLOSibilities…
Acknowledgments: We would like to thank Emma Finlayson-Trick and Dr. Craig McCormick for their time and precious advices regarding the redaction of this article. We address our gratitude toward the whole iGEM competition, our school Sup Biotech and our principal investigator, Dr. Alexandre Ismail for making this project possible and a fruitful and enriching experience.
Summary written by: iGEM IONIS team 2017
To read the full article, please click the following link: https://www.biorxiv.org/content/early/2018/03/27/289264
References :
[1] Tao Pan and Tobin Sosnick, RNA Folding During Transcription, Annu. Rev. Biophys. Biomol. Struct. 2006. 35:161–75
[2] https://food.unl.edu/escherichinia-coli-o157h7-e-coli
[3] C. Barria, M. Malecki and C. M. Arraiano, Bacterial adaptation to cold, Microbiology (2013), 159, 2437–2443
[4] Fang, L., Jiang, W., Bae, W., and Inouye, M. (1997) Mol Microbiol 23, 355-364 N.V.
[5] Bhagavan, Chung-Eun Ha, RNA and Protein Synthesis, Essentials of Medical Biochemistry (Second Edition), 2015, Pages 419–446
[6] Yamanaka K, Mitta M, Inouye M. Mutation Analysis of the 5′ Untranslated Region of the Cold Shock cspA mRNA of Escherichia coli. Journal of Bacteriology. 1999;181(20):6284-6291.
[7] Alieva NO, Konzen KA, Field SF, Meleshkevitch EA, Hunt ME, Beltran-Ramirez V, et al. (2008) Diversity and Evolution of Coral Fluorescent Proteins. PLoS ONE 3(7): e2680. https://doi.org/10.1371/journal.pone.0002680
