Plasmids 101: Repressible Promoters

Posted by Mary Gearing on May 10, 2018 9:15:54 AM


Promoters control the binding of RNA polymerase and transcription factors. Since the promoter region drives transcription of a target gene, it therefore determines the timing of gene expression and largely defines the amount of recombinant protein that will be produced. Many common promoters like T7, CMV, EF1A, and SV40, are always active and thus referred to as constitutive promoters. Others are only active under specific circumstances. In a previous post, we discussed inducible promoters, which can be switched from an OFF to an ON state, and how you might use these in your research. Today, we’ll look at repressible promoters, which can be switched from an ON to an OFF state, as well as repressible binary systems commonly used in Drosophila.

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How are repressible promoters regulated?

Like inducible promoters, repressible promoters can be regulated via positive or negative control.

Positive Repressible: Transcription is ON - an activator protein is bound and transcription is ongoing. When a repressor binds the activator protein, the activator protein cannot bind the promoter sequence anymore and transcription is turned OFF.

Negative Repressible: Transcription is ON - a repressor protein cannot bind the promoter sequence and transcription occurs unhindered. Once a co-repressor protein binds the repressor protein, the repressor protein can bind to the operator. The bound repressor then prevents transcription from occurring, which means that transcription is now OFF.

Repressible promoters Schematic-01

We’ll look at examples of both natural and engineered repressible promoters.

Chemically repressible promoters

Tet-Off

The Tet-Off system, a positive repressible promoter, was engineered from the bacterial tet operon. In the native system, the tetracycline repressor (TetR) can bind to the tetracycline operator sequences (TetO), preventing transcription. In the presence of tetracycline (Tet), TetR preferentially binds Tet and vacates the TetO elements, allowing transcription to proceed (GFP expression is controlled in this manner in pJKR-H-tetR from the Church Lab).

To turn this inducible system into a repressible system, Gossen and Bujard created the tetracycline-controlled transactivator (tTA) by fusing TetR with the transcriptional activation domain VP16. tTA binds to promoters containing TetO elements (often linked in groups of seven as a TRE), allowing transcription to proceed. When tetracycline or one of its derivatives is added, it binds tTA, removing it from the promoter and turning transcription OFF.

Tetracycline Off System Schematic

Despite their bacterial origins, Tet systems function well in mammalian cells, and TRE-containing promoters can be used in the repressible manner described above, as well as the inducible manner detailed in our previous post. For more information on Addgene Tet plasmids, see our Tetracycline resource page.

 

ADH1

The ADH1 negative repressible promoter is commonly used in yeast. In the first 24 hours of culture, when glucose is abundant and ethanol concentrations are low, pADH1 displays low promoter activity (~20% activity of the strong yeast promoter pTEF). As ethanol accumulates, it binds to the repressor, enabling it to bind the promoter and repress pADH1 activity. Researchers have engineered pADH1 variants that are less sensitive to ethanol, including the “middle” ADH1 promoter pADH1m, which maintains activity during the late ethanol consumption phase of yeast culture.

Repressible Binary Systems

GAL4/UAS

Binary systems permit exquisite control of gene expression and tracing of gene expression across development. One common binary system is the GAL4/UAS system isolated from yeast. In this system, which revolutionized genetic studies in flies, UAS basal promoter expression is low but is activated by GAL4 binding to UAS.

If you place GAL4 downstream of a tissue- or developmental stage-specific promoter and design a UAS reporter construct, the reporter will only be expressed where and/or when that promoter is active. In this way, you can interrogate the activity of uncharacterized promoters. Similarly, placing UAS upstream of a transgene permits directed expression of that gene in cells that also express GAL4.

The GAL80 repressor can partially inhibit GAL4 binding to UAS, adding a repressible element to this binary system. One can therefore place GAL4 and GAL80 under different promoters and create sophisticated patterns of UAS-driven gene expression. To further increase the complexity of the system, researchers have used split GAL4 architecture or combined the system with Flp/FRT, as reviewed by del Valle Rodriguez et al.

GAL4 UAS Binary Systems-01

LexA/lexAop

Lai and Lee developed LexA/lexAop, a complementary system to GAL4/UAS that functions in essentially the same manner.  The LexA DNA-binding domain recognizes the lexAop sequence and GAL4 or VP16 activation domain fusions to the LexA DBD result in both GAL80-repressible and GAL80-insensitive activators of lexAop. This system is commonly used with GAL4/UAS to examine the expression of reporter genes, or to combine reporter expression from one promoter with transgene or siRNA expression from the other.

The Q system

Potter et al. from Liqun Luo’s lab developed the Q system from a gene cluster in the fungus Neurospora crassa. The QUAS promoter is less leaky than UAS in the basal state, and co-expression of the inducer QF can increase QUAS driven expression by ~3,300-fold in Drosophila cells. Co-expression of repressor QS along with QF leads to intermediate expression from QUAS, as seen with GAL4/GAL80 and UAS. QF-mediated repression is reversible by the addition of quinic acid to Drosophila cells. Work by Wei et al. from Kang Shen’s lab has also shown that the Q system is also functional in C. elegans.

Subsequent work by the Luo and Potter labs has refined the Q system and introduced the second generation QF2 activators. These activators solve the problem of broad QF expression-induced lethality in flies. Riabinina et al. also developed chimeric transactivators GALQF and LexAQF, which activate their respective UAS and lexAop sequences but are also regulated by QS and quinic acid.

References

Pfeiffer, Barret D., et al. “Refinement of Tools for Targeted Gene Expression in Drosophila.” Genetics 186(2) (2010): 735-55. PubMed PMID: 20697123PubMed Central PMCID: PMC2942869.

Potter, Christopher J., et al. “The Q system: a repressible binary system for transgene expression, lineage tracing, and mosaic analysis.” Cell 141(3) (2010): 536-48. PubMed PMID: 20434990. PubMed Central PMCID: PMC2883883.

Wei, Xing, Christopher J. Potter, Liqin Luo & Kang Shen. “Controlling gene expression with the Q repressible binary expression system in Caenorhabditis elegans.” Nat Methods 9(4) (2012): 391-5. PubMed PMID: 22406855. PubMed Central PMCID: PMC3846601.

Riabinina, et al. “Improved and expanded Q-system reagents for genetic manipulations.” Nat Methods 12(3) (2015): 219-222. PubMed PMID: 25581800. PubMed Central PMCID: PMC4344399.

Lai, Sen-Li & Tzumin Lee. “Genetic mosaic with dual binary transcription systems in Drosophila.” Nat Neurosci 9(5) (2006): 703-9. PubMed PMID: 16582903.

del Valle Rodriguez, Alberto, Dominic Didiano & Claude Desplan. “Power tools for gene expression and clonal analysis in Drosophila.” Nat Methods 9(1) (2011): 47-55. PubMed PMID: 22205518. PubMed Central PMCID: PMC3574576.

De Boer, Herman A., Lisa K. Comstock & Mark Vasser. “The tac promoter: a functional hybrid derived from the trp and lac promoters.” Proc Natl Acad Sci U S A. 80(1) (1983): 21-5. PubMed PMID: 6337371. PubMed Central PMCID: PMC393301.

Da Silva, Nancy A. & Sneha Srikrishnan. “Introduction and expression of genes for metabolic engineering applications in Saccharomyces cerevisiae.” FEMS Yeast Res. 12(2) (2012): 197-214. PubMed PMID: 22129153.

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