IGEM:Melbourne/2008/BCRiboswitch/Riboswitch used in Bioclock
RNA-based riboswitches acting at the level of translation
The type of riboswitches we will be using in the Bioclock will use a short piece of RNA (called taRNA...see below) as the 'key'. In the locked, but 'ready to go' state, the gene will be transcribed, but not translated until production of the key which will allow translation.
- crRNA (cis repressed RNA): mRNA of interest containing inserted cr (cis repressive sequence). cr is a nucleotide sequence placed on the 5’UTR (untranslated region) of the gene of interest such that it is downstream of the promoter for the gene but upstream of the RBS. Its sequence should show reverse complementarity to the RBS so that under normal circumstances, the RBS is not exposed and hence translation is repressed (since the ribosome can’t bind). In addition, the mRNA will need to contain a short nucleotide sequence between cr and RBS to actually form the loop. The loop must contain a YUNR (pyrimidine uracil nucleotide purine) sequence embedded in it. Shown below is just a crude pictorial representation of what crRNA should look like:
- taRNA (trans activating RNA): This is the “key” to unlock translation. It is a piece of RNA produced under another promoter. It will also contain an RBS so to prevent its translation (so that it remains as RNA), the RBS containing sequence is sequestered in the taRNA stem structure. The 5’ region of taRNA recognises the YUNR sequence on the loop of crRNA and binds to it, causing unravelling of the crRNA. Once the RBS on the mRNA of interest is exposed, it can be translated.
With the most efficient riboswitches, it is possible to get >96% repression.
There will be targeted degradation of double-stranded RNAs by RNases. So the amount of mRNA (crRNA) available for translation itself will be reduced (since, it will have a loop structure too). Isaacs et al. were able to introduce mismatches in taRNA to protect it from RNase III cleavage.