Synthetic tools for controlling protein expression

August 26, 2010 § Leave a comment

A recent paper from Jim Collins’ lab (Callura et al. 2010, Tracking, tuning, and terminating microbial physiology using synthetic riboregulators. Proc Natl Acad Sci USA PMID: 20713708) explores the utility of a translational riboregulator to control protein production in four different settings.  The basic idea is that you set up your gene of interest so that the mRNA is produced in an inactive state, with the ribosome binding sequence hidden by an RNA hairpin; in other words, it’s cis-repressed.  Then you can control translation using a second RNA that’s complementary to the hairpin sequence, out-competing the cis-repressive sequence to release the ribosome binding sequence.

In this paper they use this system in E. coli to induce the expression of an iron transport protein, TonB, and show that they can control the expression of the TonB by changing the concentration of the activating RNA, using a tetracycline-inducible promoter.  This allows TonB to be put under dual control: the coding RNA can be expressed under an iron-sensitive promoter (as is the endogenous gene), but suppressed until triggered by the activating RNA.

Next, they showcase the predicted tight control of protein expression in this system.  It can be hard to examine the effects of expressing a toxin in a cell; cells trying to express a toxin usually die of it. Callura et al. expressed the gene encoding the toxin CcdB, which poisons DNA gyrase, under the control of the hairpin regulator.  They were then able to trigger the expression of CcdB at a defined time, allowing them to look at the transcriptional response to expression of the toxin and compare this response in two different bacterial strains, one wild-type and the other a mutant that lacks the SOS DNA repair response.

In the third application, Callura et al. use the activating RNA as a rheostat to control the amount of expression of a mutant transcription factor, Lex3A, that suppresses the SOS response.  The activating RNA is expressed under the control of an IPTG-inducible promoter.  This allowed them to partially block the SOS response, making the Lex3A-expressing cells more sensitive to the antibiotic norfloxacin than wild-type — but less sensitive to normal Lex3A-expressing cells.  The authors suggest that this kind of “tinkering” with the response of a system could be useful in testing the robustness of a network to perturbations such as changes in the timing of the expression of a component, or changes in the level of expression.

Finally, Callura et al. set out to test what is probably the “killer app” for this system, the ability to use it to control the expression of more than one gene at a time.  The number of orthogonal riboregulator sets you can make using this one basic principle is very large.  Can one combine the effects of multiple riboregulators in a predictable way, for example to make a system in which two riboregulators acting together can kill a cell?  To address this question the authors turned to the venerable lambda phage lytic system; not, this time, to study decision-making in the system (as Jacob and Monod did, and many others are still doing) but to use it as a test for independent, simultaneous regulation.  When lambda triggers a lytic event, three genes work together to cause lysis: λR, λRz and λS. Two of these, λR and λRz, work together to degrade peptidoglycan; λS makes the inner membrane of the cell permeable, and is required to get λR and λRz to where they can do their job.  So, by putting λR and λRz under the control of one riboregulator, and λS under the control of a riboregulator with different inhibition/release sequences, you should be able to make a system in which death depends on the expression of both of the trans-activating RNAs.  And they do, and it does.

The system seems to me to be analogous to a reverse form of siRNA — instead of adding small RNAs to block gene expression, you add (or cause to be expressed) small RNAs to turn on gene expression.  [Maybe — I’m sure the authors have thought of this — one could add a further riboregulator to turn expression off again.  Perhaps that’s next.]  In any case, as the authors say, this platform as it stands should have many uses.

Callura JM, Dwyer DJ, Isaacs FJ, Cantor CR, & Collins JJ (2010). Tracking, tuning, and terminating microbial physiology using synthetic riboregulators. Proceedings of the National Academy of Sciences of the United States of America PMID: 20713708

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