Activatory Switches
Introduction
Our riboswitch designs were made to regulate the expression of a downstream gene by using RNA folding to control access to a specific sequence called a ribosome-binding site (rbs). A ribosome must have access to this site to begin translation.
Some were designed to open/close co-operatively in response to the addition of two or more copies of complementary trigger strands whilst others were intended to act as controls or to help us understand various aspects of co-operativity.
Our activatory switches use a stemloop structure to hide an rbs sequence to prevent it being recognised by a ribosome. As the trigger binding sites overlap with the stem the binding of triggers to the loop should melt the stem exposing the rbs.
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Homotropic Symmetrical 8
This switch was based upon the cooperative molecular beacon by Plaxco et al. Their sequence had triggers of length 14nts, an 8bp GC-rich stem and a loop of 24nt meaning that each trigger overlapped into the stem by two nucleotides. The sequence of the stem was dictated by the sequence of the rbs (AGA GGA GA) which is then followed by six nucleotides then initiator AUG codon. This rbs sequence was used in the Yin et al. toehold switches.
We tested this switch in vivo and in vitro as a molecular beacon.
This can be considered our standard cooperative design and it defined our 1* sequence used elsewhere.
Closed
Open
Single binding site
Using the same trigger sequence as the homotropic symmetrical switch we made a switch with only a single trigger binding site. This binding site overlaps by 2 nucleotides with only the first/5’ half of the 8bp stem. The loop is 12nt, half the size of the homo sym.
This switch is expected to be non-cooperative. Our hypothesis is that the Hill coefficient for the switch will be 1. We also tested this switch in vivo and in vitro as a molecular beacon.
Heterotropic Symmetrical 8
We wanted to better understand cooperativity at a molecular level so designed a switch with two different binding sites of equal length. The 5’ binding site (bs1) is the same sequence as in the homotropic and single binding site switches so uses the same trigger (1*) whilst the second binding site (bs2) uses a different trigger (2*).
When each trigger is added alone the switch should be non-cooperative but when added
together we hypothesise they will bind cooperatively to produce a Hill curve with n>1. We tested this switch extensively in vitro where we could easily control the concentration of the two trigger strands independently. For in vivo testing, our ability to express the two triggers separately was limited by our maximum construct size and our lack of a third inducible promoter. We therefore expressed both triggers as a combined RNA separated by a TPP-inducible Hammerhead ribozyme. When thiamine is added externally it is absorbed by E. coli and converted to TPP which activates the ribozyme separating the two triggers into separate strands.
Homotropic Symmetrical 12 and 18
As all of the riboswitches found in nature are a lot larger than our designs we wanted to design a much larger cooperative switch just in case the rest of our designs are too unstable or too easily unwound by ribosomes inside cells. Homo Sym Long has a stem length of 21bp and a loop length of 32nts. It is opened by two identical triggers of length 20nt that overlap by 4nt into the stem. Both the entire rbs and the initiator AUG codon are contained within the stem resulting in a very low predicted translation rate using the Salis rbs calculator.
This switch was only tested in vivo.
Homotropic Symmetrical Long
Asymmetrical homotropic switch
We wanted to investigate the effect of lengthening the stem on cooperativity. These switches are identical to homo sym 8 but have stems of 12bp and 18bp respectively. The position of the rbs within the stem is preserved i.e. still at the bottom of the stem with the final GA sticking out.
These switches were only tested in vivo.
This switch is similar to homo sym 8 in that it has a stem length of 8 bp and two identical 14 nt trigger binding sites. It differs in that the first/5’ trigger binding site overlaps with the stem by 4nt whilst the second/3’ overlaps by only 2nts. The loop size is 22nts.
We hypothesise therefore that the 3’binding site would be used by trigger before the 5’ site.
This switch was only tested in vivo.
RBS hidden
RBS accessible
bs 1
bs 1
bs 1
bs 1
1*
1*
bs 1
bs 1
1*
bs 1
bs 2
bs 1
OR
bs 1
1*
bs 2
2*
bs 1
bs 2
1*
2*
RBS hidden
RBS accessible
Stem of 12bp
bs 1
bs 1
bs 1
bs 1
Stem of 18bp
Stem of 8bp
Stem of 8bp
Stem of 8bp
Stem of 21bp
RBS hidden
Initiator Met codon hidden
RBS and initiator Met codon accessible
bs Long
bs Long
bs Long
bs Long
Trig Long
Trig Long
Stem of 8bp
5' bs Asym
3' bs Asym
5' bs Asym
3' bs Asym
Trig Asym
Trig Asym
Closed
Open
Closed
Open
Closed
Open
Closed
Open
1* Trigger
1* and 2* Triggers
Trig Long x2
Trig Asym x2
1* Trigger x2
To investigate how increased penetration into the stem of the switch affected cooperatively we designed new triggers for this switch that go a total of 5nt's into the stem (3 more than the normal 1* trigger). This means that 1* must be elongated by 3nt's on each side. As the extra nucleotides 5' and 3' are complementary to each other, being defined by the sides of stem, when the first trigger binds to a switch the three unpaired nucleotides at the top of the loopmay act as an additional site for the recruitment of the second trigger. As this would make the binding of the second trigger easier we hypothesised that this could lead to a greater Hill coefficient than the regular 1* trigger. We called these strands "Vrigg's" (two letters onwards from Trigg).
"Vrigg"
Homo sym 8 switch
1*
extra nts
extra nts
bs1
bs1
Elongated bs
Elongated bs
1*
bs1
bs1
These three nt's can act as an additional recruitment site for another Vrigg
"Vrigg"
"Vrigg"
