Post-transcriptional Regulation Technologies - Erin Zwack

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Post-transcriptional Regulation Technologies

Overview:

Regulation of translation provides an excellent tool for research on metabolic and other pathways in organisms, and for the production of different sensors by controlling the translation of specific genes depending on cellular conditions. Further development of these types of technologies could provide a “knock-down” equivalent to RNAi, which exist in some eukaryotes. A gene of interest could be expressed normally at all times when the regulator is not active; thus, no ill effects will result before a particular pathway activates and produces a specific ligand if the gene has another purpose as well. Other synthetic biologists could use these technologies to engineer fast-responding, RNA-based biological sensors for environmental chemicals, or novel pathways.

Using RNA regulatory molecules instead of regulatory proteins to control gene expression provides several benefits to synthetic biologists. Regulatory proteins bind to specific sites such as sites on the promoter or sites upstream of the promoter called operators (wikipedia). Control by these proteins can rely heavily on cooperativity (Gardner et al. 2000), that is multiple proteins binding to one site, in order to see an effect. Regulatory RNA molecules on the other hand need a one to one ratio of regulatory molecule to target. As long as the molecule is expressed in an equal or greater amount than the target, the regulatory RNAs will normally be able to bind to their targets and control transcription.

While synthetic biologists could use the regulatory proteins and their binding sites that are found in nature, rational design of more new regulatory proteins is difficult. With the oligo and gene sythesis technology in existence today, RNA can be engineered that complements and thus targets any other RNA sequence. Regulatory proteins are also controlled mainly by promoter when determining whether they are active or not. As only so many inducible promoters exist, there is a small number of stimuli that can be used to determine under what conditions the gene under a regulatory protein's control will be expressed or repressed. An aptamer, RNA sequence that binds to a small molecule such as theophylline (Bayer and Smolke 2005), can be used not only to regulate gene expression but can also regulate under what conditions an RNA regulatory molecule is active. New aptamers are easily developed through rational design, and the number in existence is continually increasing and providing new molecules that can act as ligands.

Finally, regulatory proteins stop gene expression before transcription. When the stimulus changes and the gene is expressed (either because a regulatory protein has now bound to or has released its site), the time it takes for the phenotype to be expressed is longer because both transcription and translation must occur instead of just translation. With RNA, the gene expression is halted after transcription. Once the stimulus is removed, the RNA already produced by the gene simply needs to be translated.

Development of Systems

In most cases, post-transcriptional regulatory mechanisms that were developed and worked in eukaryotes cannot be directly transferred to prokaryotes. Modifications are necessary because eukaryotic and prokaryotic transcription and translation do not follow the exact same path. In eukaryotes, mRNA must have introns spliced out before translation begins; thus, any mechanism that regulates translation has time to bind or manipulate the mRNA (figure 1). In prokaryotes, translation begins as soon as the ribosomal binding site (RBS) is transcribed and accessible to a ribosome (figure 2).

Eukaryotic.jpg

Figure 1: Inside of the nucleus of the eukaryote, the gene is transcribed into pre-mRNA, which contain both introns (orange) and exons (red). The pre-mRNA is then modified so that the introns are spliced out and the exons are put together. Finally the mRNA are translated by the ribosome in the cytoplasm.


Prokaryote.jpg

Figure 2: In a prokaryotic cell the DNA is transcribed by RNA polymerase. As soon as the polymerase transcribes the ribosomal binding site, a ribosome binds and begins translating the sequence into protein. There is no modification step between transcription and translation.

Eukaryotes:

Antiswitches

Prokaryotes:

Riboregulators


Riboswitches

Use of Post-transciptional Regulatory Technologies

Regulation of translation provides an excellent tool for research on metabolic and other pathways in organisms, and for the production of different sensors by controlling the translation of specific genes depending on cellular conditions. Researchers can turn off translation of certain genes in response to different pathways being activated, such as metabolic pathways. If a researcher wanted to know if a particular gene were necessary to proper function of a pathway, the aptamer of the antiswitch or riboswitch could be designed to have a molecule produced upstream in the pathway be its ligand. The gene would be expressed normally at all times when the pathway is not active; thus, no ill effects will result before the pathway activates if the gene has another purpose as well. This would provide a “knock-down” equivalent to RNAi, which exists in some eukaryotes.

Synthetic biologists could engineer fast-responding, RNA-based biological sensors for environmental chemicals. The sensors could be used to detect harmful chemical like arsenic or chemical indicative of explosives by using post-transcriptional regulatory technologies that would activate or be produced in the presence of the chemical and turn-on a reporter gene like GFP. The technology could also be used to engineer novel pathways that only activate when the environmental conditions are favorable. By regulating when a newly engineered pathway is on, biologists could possibly achieve optimal efficiency in generating a desired product by not taxing the cells when they do not have enough resources to thrive and produce a product that is not natural to the organism.

The Labs

Smolke

Collins

Dr. Isaacs's Review of RNA Synthetic Biology

Gallivan

References

Bayer TS and Smolke CD. Programmable ligand-controlled riboregulators of eukaryotic gene expression. Nat Biotechnol. (2005) 3:337-43.

Desai SK and Gallivan JP. Genetic Screens and Selections for Small Molecules Based on a Synthetic Riboswitch That Activates Protein Translation. J. Am. Chem. Soc.(2004) 126:13247-54.


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.[1]

Regulatory Proteins. Wikipedia. December 2007. http://en.wikipedia.org/wiki/Gene_regulation#Regulatory_protein

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