CH391L/S12/Protein regulators of transcription

Overview

Some genes ("housekeeping genes") are expressed at a constant rate throughout the organism. This is called constitutive gene expression. More often, however, the rate at which genes are expressed changes in response to signals--this is the principle behind regulated gene expression. If the rate of gene expression increases in response to a signal, the gene is inducible; if it decreases in response to a signal, it is repressible. Repressible and inducible are not mutually exclusive, as long as the signals used are unique.

Transcription initiation is regulated by three types of proteins: specificity factors, repressors, and activators.

Specificity factors

Specificity factors facilitate RNA polymerase binding to specific promotors.

In prokaryotes

In prokaryotes, a key example of specificity factors is the σ subunit in bacterial RNA polymerase. For housekeeping genes, the σ70 subunit recognizes its specific -35 and -10 sequences in the promotor. However, in response to heat stress, the σ32 subunit replaces the σ70 subunit, and recognizes entirely different promotors. This enables certain gene products to only be expressed when the bacteria is under heat stress.

In eukaryotes

In eukaryotes, general transcription factors (GTFs) such as the TATA-binding protein (TBP) are examples of specificity factors.

Repressors: negative regulators

Molecular detail of LacI binding to the lac operator. The critical helix-turn-helix motif is highlighted in yellow. Xray crystal structure from PDB ID 1LCC.[1]

The fundamental mechanism by which repressors reduce gene expression is by blocking RNA polymerase from binding to the promotor, or blocking it from proceeding past the promotor. In prokaryotes, the sites where repressors bind (operators) are adjacent to or even overlapping with the promotor. In eukaryotes, the promotor may be quite distant from the repressor-binding site.

Repressible vs. inducible operons

The difference between repressible and inducible operons lies in the role of the effector, or molecular signal that causes a conformational change in the repressor. The effector can be either a corepressor or inducer.

If transcription is usually on but can be repressed by the binding of a corepressor to the normally inactive repressor, the operon is referred to as a repressible operon. Transcription will decrease when the repressor is activated. Repressible operons are often used in anabolic pathways, because the end product can be a feedback inhibitor of transcription. The trp operon is an example of a repressible operon, with tryptophan as the corepressor.

If transcription is normally off but can be induced by the binding of an inducer to the normally active repressor, the operon is referred to as an inducible operon. In this way, the repressor itself is "repressed", and transcription increased. Inducible operons are often used in catabolic pathways, because the enzymes involved in catabolism do not need to be synthesized unless their substrates are present. The lac operon is an example of an inducible operon, with allolactose as the inducer.

Activators: positive regulators

In contrast to repressors, activators typically lie to the 5' end of an operon's promotor (in eukaryotes, they can be quite distant to the promoter, in which case they are termed enhancers.) They facilitate RNA polymerase binding to the promotor, increasing the rate of expression. As such, they are an example of positive gene regulators.

Like repressors, activators can either be normally inactive (relying on a coactivator to become active) or active, but either way the activator is considered a positive regulator.

All-or-none induction

The expression of ara and lac follows the "all-or-none" pattern. That is, when inducer is present in nonsaturating amounts, some cells are completely induced and others remain completely repressed. If you look at a population of cells at a time, the rate of induction will appear to be linear, but this is a deceptive artifact of averaging.

This pattern is a consequence of the genes for importing inducers being a part of the operon itself. A cell may uptake an inducer, and then that inducer makes the cell drastically more likely to uptake more of the inducer; this forms a positive feedback loop and any cell induced in this manner will reduce the amount of inducers available to other cells.

Often, this doesn't matter, because addition of a saturating amount of inducer will ensure virtually all cells will be induced. However, if the inducer is toxic in high concentrations--as is the case with IPTG--all-or-none becomes an issue.[2]

More information can be found here.

lac (lactose) operon

The lac operon.[3]

Ideally, the enzymes necessary for lactose catabolism should only be synthesized when lactose is present (because otherwise, energy is wasted in unnecessary enzyme synthesis) and when glucose concentration is low (because glucose is preferentially metabolized over lactose).

To accomplish this, the lac operon makes use of both positive and negative regulation. By using both activators and repressors, transcription of the lac operon is dependent on two variables: [cAMP] (indicative of low glucose concentration) and [allolactose] (an isomer of lactose). The more variables handled, the more specifically tailored a response can be.

The lac repressor itself, LacI, is subject to constitutive regulation, as it is not part of the lac operon.[3] If LacI is not present, the lac's promoter is constitutively on.

ara (L-arabinose) operon

The ara operon is analogous to lac. Where lac has the structural genes lacZYA, ara has araBAD, which code for enzymes that catabolize arabinose into D-xylulose-5-phosphate. The initiator region, araI, consists of a promoter and operator, to which the inducible activator coded by araC binds. The inducer is arabinose (analogous to allolactose). If araC is missing (perhaps due to mutation), the ara promoter is constitutively off; this is in direct contrast to the lac operon. This difference is significant because it confers differential susceptibility to mutation.

Just as E. coli maximizes efficiency by only synthesizing the enzymes for lactose catabolism if both lactose is present and [glucose] is low, it also only expresses the enzymes for arabinose catabolism if arabinose is present and [glucose] is low. Unlike the lac operon, which achieves this via dual positive and negative regulation, the ara operon makes use of dual positive regulation (two activators). The activators in this case are CRP bound by the coactivator cAMP (identical to the lac activator), as well as AraC bound by the inducer arabinose[4]

Decoupling of the araE transporter from the inducer removes the all-or-nothing effect.[2]

Mitigation of all-or-nothing effect

Khlebnikov et al. switched control of the araE transporter from being arabinose-induced to IPTG-induced. This decoupling removed the all-or-nothing effect, because the transporter was no longer part of the ara operon.[2]

gal (galactose) operon

The repressors of gal (galactose metabolism) and mgl (methylgalactoside, glucose, and galactose transport), GalR and GalS (Gal repressor and isorepressor, respectively), act by dimerizing and creating a loop in the DNA, repressing the promoter galP1. GalR binds strongly to the external operator (O_E) of galP and weakly to mgl's O_E; the reverse is true for GalS. While gal is strongly repressed by GalR, it is weakly repressed by GalS[5]

While galR is constitutively expressed, expression of galS is dependent on CRP much like the lac and ara operons.[6]

Other ways to control expression

Tetracycline-controlled expression

Tet-On and Tet-Off systems can be used to control transcription of a gene of interest. "On" and "off" refers to whether a gene is induced by the presence ("on") or absence ("off") of doxycycline, an analogue of tetracycline.

In a Tet-Off system, the tetracycline repressor, TetR, is fused to an activation domain (VP16), which then binds to a 'tet'O operator, activating transcription. If doxycycline is present, this fused protein (tTA--tetracycline transactivator) will bind doxycycline, which causes a conformational change and prevents it from binding to the operator.

Tet-On systems, on the other hand, are only activated if doxycycline is present, because binding doxycycline allows tTA to bind the operator.

Copper-controlled expression

The ACE1 (activating copper-metallothionein expression) transcription-binding factor binds activates transcription at high [Cu²⁺] due to conformational change. The gene of interest must be downstream of a chimeric promoter that is a linkage of the CaMV 35S RNA promoter and the ACE1 transcription factor-binding site.[7]

m-toluic acid-controlled expression

The regulator XylS causes increased transcription, so it is a positive regulator (in contrast to the negative regulators LacI and AraC). Addition of m-toluic acid induces XylS dimerization, which increases the affinity for the Pm operator.[8]

Propionate-induced expression

The prpBCDE operon codes for the catabolism of propionate. Like lac and ara, it requires cAMP-bound CBP to rise above the basal rate of expression.

The main advantage of a propionate-inducible expression system as a synthetic biological tool is the fact that it does not adhere to the "all-or-none" induction model. This is due to the inducer's ability to freely diffuse across the membrane, in contrast to the transporter requirement of lac and ara. So, regulation of gene expression can occur at the cellular level instead of at the cell population level.[9]

Pragmatics

Chemoinduction

The parts necessary for an example inducible promoter to work.[10]

Imperial College's 2009 iGEM project illustrates how inducible promoters can be used to tweak gene expression levels.

When assembling a system that will result in gene expression, it is often a good idea to use an inducible rather than constitutive promoter so that gene expression is regulatable (e.g., one might only want gene expression after a population of cells is established so that they are not placed under premature energetic burden).[10]

Chromatin remodelling

Two other methods of transcription regulation via proteins include DNA methylation and histone modification.

DNA methylation

The nitrogenous bases (especially cytosine) in DNA can be methylated, affecting the rate of transcription. Methylation usually results in negative regulation, but it can alternatively result in positive regulation.

Methylation is a critical part of cell differentiation.

Histone modification

Histone acetyltransferases (HATs) catalyze the addition of acetyl groups to histone tails, causing a looser chromatin structure. This is due to the neutralization of positive charges that hold neighboring nucleosomes together. This causes the DNA to be more accessible to RNA polymerase, so it is a form of positive regulation. Conversely, histone deacetylases (HDACs) revert the histone tails to their deacetylated form, bringing neighboring nucleosomes together and making the region transcriptionally inactive.

Histone tails can also be modified by methylation or phosphorylation.

References

1. Chuprina VP, Rullmann JA, Lamerichs RM, van Boom JH, Boelens R, and Kaptein R. Structure of the complex of lac repressor headpiece and an 11 base-pair half-operator determined by nuclear magnetic resonance spectroscopy and restrained molecular dynamics. J Mol Biol. 1993 Nov 20;234(2):446-62. DOI:10.1006/jmbi.1993.1598 |

   Structure of the complex of lac repressor headpiece and an 11 base-pair half-operator determined by nuclear magnetic resonance spectroscopy and restrained molecular dynamics.

2. Khlebnikov A, Risa O, Skaug T, Carrier TA, and Keasling JD. Regulatable arabinose-inducible gene expression system with consistent control in all cells of a culture. J Bacteriol. 2000 Dec;182(24):7029-34. DOI:10.1128/JB.182.24.7029-7034.2000 |

   Regulatable arabinose-inducible gene expression system with consistent control in all cells of a culture.

3. Wilson CJ, Zhan H, Swint-Kruse L, and Matthews KS. The lactose repressor system: paradigms for regulation, allosteric behavior and protein folding. Cell Mol Life Sci. 2007 Jan;64(1):3-16. DOI:10.1007/s00018-006-6296-z |

   The lactose repressor system: paradigms for regulation, allosteric behavior and protein folding.

4. Ogden S, Haggerty D, Stoner CM, Kolodrubetz D, and Schleif R. The Escherichia coli L-arabinose operon: binding sites of the regulatory proteins and a mechanism of positive and negative regulation. Proc Natl Acad Sci U S A. 1980 Jun;77(6):3346-50. DOI:10.1073/pnas.77.6.3346 |

   The Escherichia coli L-arabinose operon: binding sites of the regulatory proteins and a mechanism of positive and negative regulation.

5. Geanacopoulos M and Adhya S. Functional characterization of roles of GalR and GalS as regulators of the gal regulon. J Bacteriol. 1997 Jan;179(1):228-34. DOI:10.1128/jb.179.1.228-234.1997 |

   Functional characterization of roles of GalR and GalS as regulators of the gal regulon.

6. Weickert MJ and Adhya S. The galactose regulon of Escherichia coli. Mol Microbiol. 1993 Oct;10(2):245-51. DOI:10.1111/j.1365-2958.1993.tb01950.x |

   The galactose regulon of Escherichia coli.

7. Mett VL, Lochhead LP, and Reynolds PH. Copper-controllable gene expression system for whole plants. Proc Natl Acad Sci U S A. 1993 May 15;90(10):4567-71. DOI:10.1073/pnas.90.10.4567 |

   Copper-controllable gene expression system for whole plants.

8. Domínguez-Cuevas P, Marín P, Busby S, Ramos JL, and Marqués S. Roles of effectors in XylS-dependent transcription activation: intramolecular domain derepression and DNA binding. J Bacteriol. 2008 May;190(9):3118-28. DOI:10.1128/JB.01784-07 |

   Roles of effectors in XylS-dependent transcription activation: intramolecular domain derepression and DNA binding.

9. Lee SK and Keasling JD. A propionate-inducible expression system for enteric bacteria. Appl Environ Microbiol. 2005 Nov;71(11):6856-62. DOI:10.1128/AEM.71.11.6856-6862.2005 |

   A propionate-inducible expression system for enteric bacteria.

10. http://2009.igem.org/Team:Imperial_College_London/Chemoinduction

    2009 iGEM team with an inducible promoter