Difference between revisions of "Team:Exeter/RNA Riboswitches"

 
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        <li class="level_3"><a onclick="$('.Basic, .Trans, .RNA, .Toehold').hide('slow');$('.Ribo').toggle('slow')">
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            General Riboswitches
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        <li class="level_3"><a onclick="$('.Basic, .Trans, .RNA, .Ribo').hide('slow');$('.Toehold').toggle('slow')">
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            Synthetic Toehold Switches
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<h1>RNA and Riboswitches</h1>
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<p style = "font-size: 20px; color: #608341"><b><em>"In the beginning, RNA was a simple molecule, but over time it has gained many functions. From self-replication, to storing and utilising information, to regulating cellular pathways, it is an example to all molecules..."</em></b></p>
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    The Central Dogma
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    <li class="previous"><a href="https://2015.igem.org/Team:Exeter/Fundamentals"><span aria-hidden="true">&larr;</span>Fundamental information</a></li>
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<p>For more fundamental background information, <a href="https://2015.igem.org/Team:Exeter/Fundamentals">click here</a>.
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    The RNA molecule
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<h2>Riboswitches</h2>
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<p>
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Riboswitches are essentially mRNA molecules which have a regulatory section which controls whether or not the protein coding section is read or not. There are quite a few different types of riboswitches, each with different mechanisms. In general, the presence or absence of a trigger (from small metal ions, amino acids, proteins and other RNA molecules), or changes in conditions such as temperature and pH, causes a change in conformation of the riboswitch which then either allows or stops the protein coding region from being read by a ribosome and the protein produced. Broadly, riboswitches can be put into two groups; those which act at the transcriptional level, and those at the translational level. This section will describe the general mechanisms of each type.
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<h4>Transcriptional Control:</h4>
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<div class="slide"><a href="https://static.igem.org/mediawiki/2015/9/96/Exeter_transcription_riboswitch.png"><img src="https://static.igem.org/mediawiki/2015/9/96/Exeter_transcription_riboswitch.png" title="Figure 1: Transcription-level riboswitch."></a></div>
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One of the main types of riboswitches controls the production of its own mRNA. During transcription, the riboswitch section of the mRNA is produced first while the rest of the coding section is being synthesised. This regulatory section is able to take on one of two structures depending on the presence/absence of a ligand or change in conditions.</br>
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If the riboswitch is turned off by either the conditions or ligand presence, then it will cause a terminator to form in the mRNA and hence the RNA polymerase will stop transcription before the entire mRNA is produced, resulting in an essentially useless mRNA molecule. However, if the riboswitch is turned on, then an anti-terminator is formed instead and the RNA polymerase is able to read through the entire mRNA and allow the protein to be expressed (Figure 1).
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<h4>Translational control:</h4>
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<div class="slide"><a href="https://static.igem.org/mediawiki/2015/c/c4/Exeter_translation_riboswitch.png"><img src="https://static.igem.org/mediawiki/2015/c/c4/Exeter_translation_riboswitch.png" title="Figure 2: Transcription-level riboswitch."></a></div>
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Riboswitches which act at the translational level usually work by sequestering the RBS (Ribosome Binding Site) away from the ribosome, and hence stop translation of the mRNA and protein production. There are different mechanisms of sequestering and revealing the RBS. One of these ways is through cleavage of the riboswitch. In the absence/presence of a certain ligand or condition, the riboswitch can take on a conformation in which a cleavage site is revealed. If the riboswitch becomes cleaved, then the RBS can be released and accessed by a ribosome, which can then read the protein coding region of the mRNA. The cleavage of this riboswitch can be carried out by a protein/ribozyme, or in some cases by the riboswitch itself.</br>
 
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<h2 class="Dogma">The Central Dogma</h2>
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Another way in which an RBS can be sequestered is by being placed within a loop structure. When in a loop structure, the ribosome is unable to bind to the RBS sequence, and hence the protein can't be produced. If a ligand/condition changes the conformation of the riboswitch causing the loop structure to be removed, then the ribosome becomes able to bind to the RBS and read the rest of the mRNA (Figure 2).
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<h4>Other types of riboswitches:</h4>
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The central dogma is used to explain how information encoded on DNA is used to create what we know as life. Essentially, DNA is converted to RNA which is converted to proteins (figure 1).</br>
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There are many types of riboswitches which are found in nature, each with a different type of mechanism. Our project is based around a specific riboswitch called a toehold switch. In the next section, the use of riboswitches in synthetic biology will be discussed in addition to a description of the structure and mechanism of a toehold switch.
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<h2>Natural and Synthetic Toehold Switches</h2>
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As has been discussed in the previous sections, RNA is an important molecule which is involved in many functions, including cellular regulation. Discussed in some detail in the previous section were riboswitches and the different types and mechanisms of action. For our project we have designed and improved upon a specific type of riboswitch; a toehold switch.</br>
 
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DNA (Deoxyribonucleic acid) is a polymer molecule (made up of sub-units called nucleotides or bases) which has a double stranded helical structure (figure 1a). There are four types of bases (Guanine - G, Cytosine - C, Adenosine - A, and Tyrosine - T)which can be joined together in many different conformations to form different DNA molecules, and each type of base is able to pair with one other type; C pairs with G, and A pairs with T (figure 2). Bases which pair are described as being complementary.</br>
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Toehold switches are riboswitches which regulate at the transcriptional level via RBS sequestration, and they are so named for the toehold structure which is an integral part of the riboswitch (figure 3). The basic mechanism of action is that an RNA molecule with a complementary sequence to that of the switch region of the toehold switch binds to the switch and causes the structure to open up, removing the toehold structure and revealing the RBS to allow the ribosome to bind. This mechanism is discussed in further detail below.</br>
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DNA's primary job is to store biological information in the form of genes, which are encoded by the bases which make up the DNA molecule. For example, CGGGATGTATTAC could encode for a specific gene.</br>
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RNA (RiboNucleic Acid) is similar to DNA in that it is a polymer molecule and made up of nucleotides/bases, but it differs in a few crucial ways. The first is that usually RNA is made up of only a single strand, as opposed to DNA's double stranded structure (figure 1b). The second is that in RNA, the Tyrosine (T) base is not used, instead it is replaced with Uracil (U).</br>
 
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As mentioned above, RNA is the second stage of The Central Dogma. Usually, a type of RNA termed mRNA is used as an intermediate between DNA and proteins and is made using DNA as a template, meaning that the sequence of the RNA molecule is determined by the sequence of the DNA from which it is copied. There are many reasons why an intermediate is required instead of simply using DNA. These reasons include:
 
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<li>Protection of the DNA: damage to DNA can cause unfavourable mutations so it is safer to use a 'copy' rather than the original,</li>
 
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<li>Regulatory reasons: the presence or absence of RNA can correspond to the presence/absence of the protein which it encodes for, meaning that it can be used to control cellular pathways</li>
 
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<li>Inability of DNA to reach protein machinery: in eukaryotic cells (animals, plants, fungi, etc.), the DNA is separated from the rest of the cell by a nuclear envelope, DNA is unable to pass through this envelope but RNA is able to</li>
 
  
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<h4>Green et al. 2014:</h4>
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Riboswitches are found in abundance naturally in bacteria and work well as regulators, however synthetic biology is in the business of taking things found in nature and adapting them to our own needs. This is exactly what a research group led by Alexander Green (<a target="_blank" href="http://collinslab.mit.edu/files/cell_green.pdf">Green <em>et al.</em> 2014</a>) has done.</br>
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Proteins are the end product of The Central Dogma and are used to carry out functions and generally create what we recognise as life. Proteins are also polymer molecules made up of subunits, but unlike with DNA and RNA these subunits are not bases/nucleotides, they are amino acids. Amino acids are relatively simple molecules which all share a generic structure, but have different functional (R) groups (figure 3). The interactions of the functional groups, both with other functional groups of the same/different proteins, and with other molecules/etc. in its environment, gives the protein its overall function. These functions can range from catalytic (speed up the rate of a reaction) to structural (shape/strength of a cell), to virulence (causing disease in a host).</br>
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As can hopefully be seen from above, RNA is a vital part of The Central Dogma, and therefore is fundamental to life.
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<div class="slide"><a href="https://static.igem.org/mediawiki/2015/5/50/Exeter_generic_riboswitch.png"><img src="https://static.igem.org/mediawiki/2015/5/50/Exeter_generic_riboswitch.png" title="Figure 4: General riboswitch structure."></a></div>
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Riboswitches have the potential to be amazingly useful tools in a range of areas (discussed in more detail on the <a href="https://2015.igem.org/Team:Exeter/Future">'Future'</a> page), however natural riboswitches have a few issues which means that they are not as easy to use as tools as they might be. One of the limitations of these riboswitches is that they tend to have a low dynamic range. The dynamic range can be thought of as the ratio between the high and low levels of a signal - in the case of riboswitches this would be the ratio between the levels of protein controlled by the riboswitch when switch is on vs. off. Typically, natural riboswitches have dynamic ranges of about 55 fold for riboswitches which enhance protein production, and about 10 fold for those which repress production. Another limitation is that natural riboswitches tend to have significant cross-talk (i.e. their activity is able to be altered by more than one input), which can make their specificity relatively low.</br>
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<h2 class="RNA">The RNA Molecule</h2>
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<div class="slide"><a href="https://static.igem.org/mediawiki/2015/4/47/Exeter_design_constraints_toehold.png"><img src="https://static.igem.org/mediawiki/2015/4/47/Exeter_design_constraints_toehold.png" title="Figure 5: A toehold structure labelled according to design."></a></div>
  
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As has been mentioned briefly, RNA is a single stranded, helically structured polymer molecule made up of nucleotides/bases. While it may seem that this structure is much simpler than DNA, the fact that it doesn't have all of its bases already paired to its complementary strand means that the RNA's nucleotides are free to base pair in many different ways. For example, the RNA molecule could base pair with itself (figure 1a) or other molecules to form a complex (figure 1b). The ways in which the RNA bases interact defines the (secondary) structure of the molecule, so therefore the sequence of the RNA molecule defines the structure of the molecule. This means that if a specific RNA structure is required, then it should be able to be achieved by giving the RNA a specific sequence. This is shown in figure 2. The RNA molecule has two sections which are complementary to each other, which can therefore base pair to create a stem region. The bases which are not complementary remain un-paired and create a loop at the top of the stem section. The fact that RNA is able to fold into many types of secondary structures means that it can have a variety of functions.</br>
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Synthetic biology is based on being able to easily engineer biological 'tools' to our own needs, however the structure of some natural riboswitches can make this difficult. Figure 4 shows a generic structure of many natural RNA-binding riboswitches. The region labelled as the 'switch region' is where the trigger RNA binds to activate the riboswitch. As can be seen, there are two regions which give constraints to the switch region sequence. The first of these are that a section near the middle of the switch region must show complementation to the RBS, the second is that the end of the region must follow the YUNR motif. The YUNR motif is a pattern of nucleotides which allows the binding of trigger RNA as either a loop-linear interaction, or a loop-loop interaction. These constraints increase the difficulty of engineering these riboswitches as trigger RNAs have the same constraints as the switch region. This can cause cross-talk between riboswitches within the same system as the triggers will have regions which show homology (the same/very similar) due to the same constraints being imposed upon them. If this issue could be worked around, then not only would it make riboswitches easier to engineer, but also reduce the issue of cross talk. In fact, this is exactly what Green <em>et al.</em> did.</br>
 
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One cellular process in which RNA is heavily involved is that of protein synthesis. We have already mentioned how RNA acts as an intermediate and contains a sequence which corresponds to the amino acid sequence of a protein, but we haven't mentioned how this happens.</br>
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The research group led by Alexander Green designed a toehold switch of the general structure shown in figure 5. As can be seen, the constraints placed upon the switch region in natural riboswitches are no longer present.</br>
 
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mRNA (messenger RNA) is able to encode for amino acids through the use of 'triplets', also known as 'codons'. These are simply three bases on mRNA which corresponds to a single amino acid, of which there are 21 (natural) types (figure 2). For example, the codon AUG codes for the amino acid methionine (M).</br>
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<h4>Toehold switch mechanism:</h4>
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The toehold switch mechanism is similar to any other RNA-binding riboswitch which regulates at the translational level. The trigger RNA binds to the switch region of the toehold in a linear-linear way, causing the toehold structure to open up. This then releases the RBS from the loop, allowing a ribosome to bind it in a linear-linear way. The ribosome can then read along the coding region of the toehold switch, hence giving off a signal. The Green <em>et al.</em> 2014 paper shows that toehold switches of this design are able to have dynamic ranges of over 400 (comparable to below 60 for natural riboswitches), and a crosstalk level of below 12%. These changes mean that toehold switches are more suitable for use in synthetic systems.</br>
 
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While codons allow mRNA to encode the amino acid sequence of a protein, they do not explain how this information is used practically. In order to do this, we must look at another type of RNA; tRNA (transfer RNA). AS can be seen in figure 3, tRNA has an interesting secondary structure, and two important regions. The first of these regions is the attachment site at the top of the tRNA, which is where a specific amino acid to the tRNA is able to attach. The second region is the anti-codon at the bottom of the molecule. The anti-codon is complementary to the codon for the amino acid which is attached to that tRNA, allowing the tRNA to bind to the mRNA, and hence ensure that the amino acid is added to the sequence in the correct place (figure 4).</br>
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<h4>Our Development:</h4>
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These toehold switches show amazing potential in many areas, but we think that the area of diagnostics could greatly benefit from the development of toehold switches. This is why we decided to develop further the toehold switches made by Green <em>et al.</em>. We plan to develop a standard toehold switch which can be changed in a relatively simple way in order to detect any given RNA, and hence diagnose many different diseases. We also wish to make a set of toehold switches with different indicators, mainly fluorescence, colour change, and luminescence. Another of our goals is to characterise toehold switches in a cell free system, and use the data from this to inform a model, which could then predict how other toeholds may act under the same conditions without carrying out many different experiments.</br>
 
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There is still one more main part of this mechanism which is missing, and that is how the amide bonds between amino acids are formed in order to synthesis the protein. Once again, RNA comes to the rescue, this time in the form of rRNA (ribosomal RNA). The are different types of rRNA, and they come together (along with some proteins) to form a specific complex called a ribosome (figure 5, also pictured in our logo). The ribosome's job is to bind to the mRNA and 'read' along it, ensuring that the correct tRNAs are added at the right time (figure 6).</br>
 
 
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Protein synthesis is just one of the many pathways and processes in which RNA is involved, in the next section we will see how RNA can help regulate cellular pathways.
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<h2 class="Regulation">RNA in Regulation</h2>
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Latest revision as of 01:38, 19 September 2015

Toehold Background

For more fundamental background information, click here.

Riboswitches

Riboswitches are essentially mRNA molecules which have a regulatory section which controls whether or not the protein coding section is read or not. There are quite a few different types of riboswitches, each with different mechanisms. In general, the presence or absence of a trigger (from small metal ions, amino acids, proteins and other RNA molecules), or changes in conditions such as temperature and pH, causes a change in conformation of the riboswitch which then either allows or stops the protein coding region from being read by a ribosome and the protein produced. Broadly, riboswitches can be put into two groups; those which act at the transcriptional level, and those at the translational level. This section will describe the general mechanisms of each type.

Transcriptional Control:

One of the main types of riboswitches controls the production of its own mRNA. During transcription, the riboswitch section of the mRNA is produced first while the rest of the coding section is being synthesised. This regulatory section is able to take on one of two structures depending on the presence/absence of a ligand or change in conditions.
If the riboswitch is turned off by either the conditions or ligand presence, then it will cause a terminator to form in the mRNA and hence the RNA polymerase will stop transcription before the entire mRNA is produced, resulting in an essentially useless mRNA molecule. However, if the riboswitch is turned on, then an anti-terminator is formed instead and the RNA polymerase is able to read through the entire mRNA and allow the protein to be expressed (Figure 1).

Translational control:

Riboswitches which act at the translational level usually work by sequestering the RBS (Ribosome Binding Site) away from the ribosome, and hence stop translation of the mRNA and protein production. There are different mechanisms of sequestering and revealing the RBS. One of these ways is through cleavage of the riboswitch. In the absence/presence of a certain ligand or condition, the riboswitch can take on a conformation in which a cleavage site is revealed. If the riboswitch becomes cleaved, then the RBS can be released and accessed by a ribosome, which can then read the protein coding region of the mRNA. The cleavage of this riboswitch can be carried out by a protein/ribozyme, or in some cases by the riboswitch itself.

Another way in which an RBS can be sequestered is by being placed within a loop structure. When in a loop structure, the ribosome is unable to bind to the RBS sequence, and hence the protein can't be produced. If a ligand/condition changes the conformation of the riboswitch causing the loop structure to be removed, then the ribosome becomes able to bind to the RBS and read the rest of the mRNA (Figure 2).

Other types of riboswitches:

There are many types of riboswitches which are found in nature, each with a different type of mechanism. Our project is based around a specific riboswitch called a toehold switch. In the next section, the use of riboswitches in synthetic biology will be discussed in addition to a description of the structure and mechanism of a toehold switch.

Natural and Synthetic Toehold Switches

As has been discussed in the previous sections, RNA is an important molecule which is involved in many functions, including cellular regulation. Discussed in some detail in the previous section were riboswitches and the different types and mechanisms of action. For our project we have designed and improved upon a specific type of riboswitch; a toehold switch.

Toehold switches are riboswitches which regulate at the transcriptional level via RBS sequestration, and they are so named for the toehold structure which is an integral part of the riboswitch (figure 3). The basic mechanism of action is that an RNA molecule with a complementary sequence to that of the switch region of the toehold switch binds to the switch and causes the structure to open up, removing the toehold structure and revealing the RBS to allow the ribosome to bind. This mechanism is discussed in further detail below.

Green et al. 2014:

Riboswitches are found in abundance naturally in bacteria and work well as regulators, however synthetic biology is in the business of taking things found in nature and adapting them to our own needs. This is exactly what a research group led by Alexander Green (Green et al. 2014) has done.

Riboswitches have the potential to be amazingly useful tools in a range of areas (discussed in more detail on the 'Future' page), however natural riboswitches have a few issues which means that they are not as easy to use as tools as they might be. One of the limitations of these riboswitches is that they tend to have a low dynamic range. The dynamic range can be thought of as the ratio between the high and low levels of a signal - in the case of riboswitches this would be the ratio between the levels of protein controlled by the riboswitch when switch is on vs. off. Typically, natural riboswitches have dynamic ranges of about 55 fold for riboswitches which enhance protein production, and about 10 fold for those which repress production. Another limitation is that natural riboswitches tend to have significant cross-talk (i.e. their activity is able to be altered by more than one input), which can make their specificity relatively low.

Synthetic biology is based on being able to easily engineer biological 'tools' to our own needs, however the structure of some natural riboswitches can make this difficult. Figure 4 shows a generic structure of many natural RNA-binding riboswitches. The region labelled as the 'switch region' is where the trigger RNA binds to activate the riboswitch. As can be seen, there are two regions which give constraints to the switch region sequence. The first of these are that a section near the middle of the switch region must show complementation to the RBS, the second is that the end of the region must follow the YUNR motif. The YUNR motif is a pattern of nucleotides which allows the binding of trigger RNA as either a loop-linear interaction, or a loop-loop interaction. These constraints increase the difficulty of engineering these riboswitches as trigger RNAs have the same constraints as the switch region. This can cause cross-talk between riboswitches within the same system as the triggers will have regions which show homology (the same/very similar) due to the same constraints being imposed upon them. If this issue could be worked around, then not only would it make riboswitches easier to engineer, but also reduce the issue of cross talk. In fact, this is exactly what Green et al. did.

The research group led by Alexander Green designed a toehold switch of the general structure shown in figure 5. As can be seen, the constraints placed upon the switch region in natural riboswitches are no longer present.

Toehold switch mechanism:

The toehold switch mechanism is similar to any other RNA-binding riboswitch which regulates at the translational level. The trigger RNA binds to the switch region of the toehold in a linear-linear way, causing the toehold structure to open up. This then releases the RBS from the loop, allowing a ribosome to bind it in a linear-linear way. The ribosome can then read along the coding region of the toehold switch, hence giving off a signal. The Green et al. 2014 paper shows that toehold switches of this design are able to have dynamic ranges of over 400 (comparable to below 60 for natural riboswitches), and a crosstalk level of below 12%. These changes mean that toehold switches are more suitable for use in synthetic systems.

Our Development:

These toehold switches show amazing potential in many areas, but we think that the area of diagnostics could greatly benefit from the development of toehold switches. This is why we decided to develop further the toehold switches made by Green et al.. We plan to develop a standard toehold switch which can be changed in a relatively simple way in order to detect any given RNA, and hence diagnose many different diseases. We also wish to make a set of toehold switches with different indicators, mainly fluorescence, colour change, and luminescence. Another of our goals is to characterise toehold switches in a cell free system, and use the data from this to inform a model, which could then predict how other toeholds may act under the same conditions without carrying out many different experiments.


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