From GcatWiki
Jump to: navigation, search

All information on riboregulators came from Isaacs et al. 2004.

After observing the many and varied naturally occuring post-transcriptional, regulatory RNA systems, Isaacs et al. designed and engineered a modular synthetic system where RNA turns on and off gene expression by controlling translation. The modularity of their system allows any gene to be regulated instead of only a specific gene to which the riboregulator is targeted.


The design itself has two components: a short cis-repressed RNA sequence (crRNA) that is inserted upstream of the gene and a transactivating RNA sequence (taRNA) that targets the crRNA and is free floating. The crRNA sequence contains two fundamental components: the complement of the ribosomal binding site (RBS) and a pyrimidine-uracil-nucleotide-purine (YUNR) sequence (figure 9). When not interacting with the taRNA, the complement of the RBS binds to the RBS, causing the crRNA to loop and block the ribosome's access to the RBS. When the RBS is blocked, translation does not occur; the gene expression is off. The YUNR sequence has a complement on the taRNA. When the taRNA finds a crRNA, the interaction with the YUNR sequence begins pulling the crRNA off the RBS (figure 11).


(Isaacs et al. 2004)

Figure 9: Cis repressing RNA, crRNA, sequence is inserted upstream of the RBS. Part of the crRNA (red) complements the RBS and a few bases on either side. This section of the crRNA is not an exact complement; thus, the crRNA can be peeled off the RBS by the trans activating RNA, taRNA. The complementary sequence to YUNR is found in the ta-RNA and begins peeling off the crRNA by binding to it (figure 11).

The taRNA is free floating and regulated by an inducible promoter. The inducible promoter allows the researcher to determine under what conditions translation will be allowed. Besides the complement to the YUNR sequence, the taRNA contains a section of high complementarity to the section of the crRNA that folds over to cover the RBS. This complementary sequence allows the taRNA to keep the crRNA sequestered from the RBS (figure 10).


(Isaacs et al. 2004)

Figure 10: taRNA when not in contact with crRNA sequesters the complement to the crRNA as it is extremely close to the RBS sequence. If the complement to the crRNA was not sequestered until the taRNA comes in contact with the YUNR sequence, ribosomes could potentially bind to the taRNA and decrease the efficiency of translation in the cell. When the YUNR complement binds to the YUNR sequence in the cr-RNA, the binding of the crRNA to the rest of its complement in the taRNA begins, and the crRNA is pulled off the RBS (figure 11).

To recap, gene expression is off when there is crRNA upstream of the gene but no taRNA is in the system. In the presence of taRNA, gene expression is turned back on.

Riboregulator system.JPG

(Isaacs et al. 2004)

Figure 11: a. the crRNA is bound to the RBS and blocks ribosomes from translating the mRNA into protein. b. the taRNA comes in contact with the YUNR sequence on the crRNA and begins to bind to the rest of the crRNA. c. the taRNA fully bound to the crRNA and peeled the crRNA off the RBS. The RBS is now free for the ribosome to bind to and begin translation.

From Concept to Wet Lab

In wet lab, the crRNA sequence was placed in front of the GFP gene and introduced to cells. Using flow cytometry, Isaacs et al. measured fluorescence of cells that had just the GFP gene, the GFP gene with crRNA upstream, the GFP gene with crRNA upstream and taRNA, and no GFP at all. The crRNA decreased fluorescence to near basal levels. When taRNA was present in the system, fluorescence increased by approximately a power of ten. While the fluorescence did not equal fluorescence of cells with just GFP, the repression of gene expression with crRNA and return of expression with taRNA are strong enough to suggest the riboregulator system works (figure 12).

Experimental taRNA.JPG

(Isaacs et al. 2004)

Figure 12: The black curve represents fluorescence in cells that do not have a GFP gene (ie. the cells natural autofluorescence). The red curve represents fluorescence in cells that have GFP with crRNA upstream. The green curve represents fluorescence in cells that have GFP with crRNA upstream and produce the taRNA. The blue curve represents the fluorescence of cells that have normal GFP minus the cells' autofluorescence.

Finally, riboregulators can control different genes in response to different stimuli by using different crRNA/taRNA pairs as the pairs are specific to each other (figure 13).


(Isaacs et al. 2004)

Figure 13: The graph shows both GFP fluorescence (black and white bars) when the taRNA promoter, pBad, is off (- arabinose) and on (+ arabinose). All data is normalized to + arabinose GFP and RNA levels. The low level GFP fluorescence when no arabinose is present shows that the efficiency of crRNA is not 100% but is still high. As high GFP fluorescence is seen only when high amounts of the matching taRNA is present and not simply when any taRNA variant is present, this data shows that riboregulator pairs are specific and multiple pairs can be used to regulate multiple genes without fear that any taRNA produced would activate all the RNAs and not just its target RNA.


While all of these experiments were done with the GFP gene, Isaacs et al. designed their riboregulator to be modular, capable of being used with any gene (Isaacs et al. 2004). As the taRNA simply targets the crRNA and the crRNA can be placed in front of any gene, it can be considered modular. The above experiments were done with pBad and pLac controlling the production of taRNA. As the system worked when the taRNA was under control of either promoter and the crRNA can be inserted upstream of any gene, this system is basically considered modular.

The only caveat is that the crRNA construct added to the gene will need to contain the RBS unless the gene's RBS is close enough to the complement to bind to it. Even small changes to a ribosomal binding site can can change the transcription rate of genes (Gardner et al. 2000). If the original RBS is not close enough to the complement in the crRNA and you desire to keep the original transcriptional rate and level, you would have to redesign the crRNA and the taRNA as the complement to the RBS in the crRNA and the complement to the crRNA in the taRNA would need to be different.

Further Work

As mentioned in the overview, using regulatory proteins or inducible promoters limits the number of stimuli (molecules) that can be used to determine when expression should occur. Generating taRNA that is ligand controlled like an antiswitch or riboswitch would provide a greater versatility for riboregulator use. Ligand controlled riboregulators may also be more effective as control by a ligand may decrease leaky activity, which occurs with promoters as they always allow for some basal level of transcription. Isaacs has continued working to engineer a ligand controlled version of the riboregulator.


Gardner, T.S., Cantor, C.R., and Collins, J.J. Construction of a genetic toggle switch in Eschreichia coli. Nature (2000) 403: 339-342.

Isaacs FJ, et al. Engineered riboregulators enable post-transcriptional control of gene expression. Nat Biotechnol. (2004) 22:841-47.

Return to Post-transcriptional Regulation Technologies