lac operon

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The lactose operon , shortly lac operon is an operon (a functional unit of the deoxyribonucleic acid ), which both during transport and in the breakdown of lactose in bacteria , for example Escherichia coli , plays an important role. The lac operon is one of the classic model systems for gene regulation; an extracellular signal (here the availability of certain sugars) is translated into an energetically favorable adaptation of the cell's metabolism by switching the structural genes of the operon on or off.

Structure of the operon

The lac operon consists of a promoter (P), three operators (O 1 , O 2 and O 3 ) and three structural genes :

  • The lacZ - gene coding for the enzyme β-galactosidase (LacZ, EC  3.2.1.23 ). It hydrolyzes, i.e. splits lactose into galactose and glucose , but can also convert lactose into allolactose , an isomer of lactose.
  • The lacY gene codes for a transport protein called β-galactoside permease (LacY), which enables the uptake of lactose into the cell .
  • The lacA gene codes for the enzyme β-galactoside transacetylase ( EC  2.3.1.18 ). It is not necessary for the breakdown of lactose and its function has not been definitively established. However, there are indications that the acetylation of non-degradable β-galactosides by the enzyme has a detoxification function for the cell.
Schematic structure of the lac operon (not to scale)

Regulation of the operon

These three genes of the lac operon are only expressed when lactose is present in the surrounding medium and there is no more favorable energy source for the cell. Such a preferable source of energy is e.g. B. glucose. A system of negative and positive regulation controls the breakdown of the most efficient energy source.

The lac operon is both negatively by a repressor, and positively by an activator regulated . In addition, the mechanism of inducer exclusion controls the activity of the lac permease. These three regulations mean that lactose can only be metabolized when there is no more efficient alternative.

Negative regulation

The negative regulation of the lac operon takes place at the transcription level by the lac repressor . The repressor is a homotetrameric protein that can bind to two of the operators at the same time: O 1 and O 2 or O 1 and O 3. The advantage of negative regulation is that, as long as no lactose has to be metabolized, there are no enzymes for it Dismantling must be provided. The actual repression takes place via the binding of the repressor to O 1 . The simultaneous attachment to two operators has the consequence that the DNA lying between the two operators forms a loop shape. This affects the strength of the repression: if the repressor can only bind to O 1 (in mutants lacking O 2 and O 3 ), the repression drops from about 1000-fold to about 20-fold. If, however, it binds either to O 1 and O 2 (in mutants that lack O 3 ) or to O 1 and O 3 (in mutants that lack O 2 ) at the same time, the genes of the operon are around factors 700 and 440 are repressed.

The reason for the drop in the expression rate is that the RNA polymerase required for this can no longer bind efficiently to the DNA. This prevents the gene from being read, the transcription .

The repressor is encoded by a regulatory gene , the lacI gene. This gene is located directly in front of the lac operon (see picture above) and is expressed by its own constitutive promoter. Since the operator O 3 overlaps with the 5 'end of the lacI gene, if this operator is bound in a DNA loop with O 1 with high affinity by Lac repressor, an impairment of the RNA polymerase can occur. It can no longer get to the end of the gene and falls off the DNA. This means that the mRNA of the lacI gene is incomplete; the stop codon , for example, is missing . If the incomplete RNA is then translated , ribosomes get stuck, since without a stop codon the usually releasing factors of termination ( release factor RF) are not effective. In the bacterial cell, such persistent ribosomes are freed by tmRNA ( trans translation) and the incomplete LacI protein is marked for degradation. This process of trans translation is an important process in the regulation of the lac operon; without tmRNA, incomplete lacI repressors would arise, some of which would still be active.

Positive regulation

An activator protein is responsible for the positive regulation of the lac operon: CAP ( catabolite activator protein ), the best-studied effector of catabolite repression . The CAP activity is directly dependent on the concentration of the cAMP . The addition of cAMP to CAP causes a conformational change in the regulatory protein, which greatly increases its specific DNA binding. The CAP-cAMP complex attaches to a binding site in the DNA and from there interacts directly with the RNA polymerase. This significantly increases the affinity of the RNA polymerase for the promoter. So three elements are necessary for this positive regulation of the lac promoter: the CAP protein, cAMP and a CAP binding site in the lac promoter. Mutations that lead either to the absence of the CAP protein or the absence of cAMP or to the inactivation of the CAP binding site have the same effect on the lac operon. Its expression drops about 50 times. Catabolite repression is absent in the lac UV5 mutant , which is why it is used for overexpression of bacterial recombinant proteins .

Influence of lactose

If lactose is the most efficient substrate around the cell as an energy supplier, it is brought into the cell by the β-galactoside permease. There it is partially converted into allolactose by β-galactosidase . This means that the Gal-β-1,4-Glc bond is converted into a Gal-β-1,6-Glc bond. In this form, an attachment to the lac repressor is possible.

This attachment changes the conformation of the repressor and it separates from the operator. The RNA polymerase can now start transcribing . The subsequent translation provides additional permease and β-galactosidase molecules. Lactose can thus be used permanently as a substrate until it is used up or a better source of energy is available.

Influence of glucose

It is advantageous for the cell to prefer glucose to lactose as a substrate. Consequently, glucose must break down before lactose. For this to happen, glucose has to delay the breakdown of lactose. However, this does not happen directly through the glucose itself. The phosphotransferase system is used to transport the glucose into the cell . The phosphate residue from PEP is transferred to the glucose via the transport protein E IIA. The unphosphorylated E IIA now inhibits the lactose permease, which means that no lactose is transported into the cell and the lac operon remains inactivated. This process is known as inductor exclusion. Even in the presence of lactose, there is hardly any gene expression and glucose is broken down preferentially.

Many school books cite a decrease in the cAMP concentration as the reason for the influence of glucose on the expression of the lac operon. However, this assumption is outdated today. It was found that the cAMP concentrations in the presence of lactose are approximately the same as in the presence of glucose or both sugars at the same time.

history

In 1961, the French scientists François Jacob and Jacques Monod developed the operon model of gene regulation based on the lac operon of Escherichia coli . For this work they received the Nobel Prize in Physiology or Medicine in 1965 .

Special features of the Lac operon

Lactose is generally absorbed in the human small intestine, but E. coli is mainly found in the large intestine. As a result, lactose is normally not available as a food source, or only in small amounts, for E. coli in the large intestine. In fact, the lactose operon serves more to break down glyceryl galactosides. These generally arise from the breakdown of fats in animal cells and, in this case, when the cells lining the colon are shed and disintegrate. Glyceryl galactosides serve here both as an inducer for the LacI protein (repressor) and as a substrate for β-galactosidase, which splits glyceryl galactosides into glycerol and galactose.

Furthermore, the lac operon cannot be detected in salmonella and various other intestinal bacteria ( enterobacteria ) that are relatively closely related to E. coli . It is likely that this segment is relatively new in the E. coli genome from an evolutionary point of view and originally arose outside the enterobacteria.

literature

Individual evidence

  1. ^ SL Roderick (2005): The lac operon galactoside acetyltransferase. In: CR Biologies Vol. 328, pp. 568-575. PMID 15950163
  2. ^ A b Santillán, M. and Mackey, MC. (2008): Quantitative approaches to the study of bistability in the lac operon of Escherichia coli . In: JR Soc Interface 6; 5 Suppl 1: S29-39; PMID 18426771 ; PDF (free full text access)
  3. ↑ Keiler, KC. (2008): Biology of trans-translation . In: Annu Rev Microbiol . 62; 133-151; PMID 18557701 ; doi: 10.1146 / annurev.micro.62.081307.162948
  4. ^ Abo, T. et al . (2000): SsrA-mediated tagging and proteolysis of LacI and its role in the regulation of lac operon . In: EMBO J . 19 (14); 3762-3769; PMID 10899129 ; PMC 313975 (free full text)
  5. ^ AE Silverstone, RR Arditti, B. Magasanik: Catabolite-insensitive revertants of lac promoter mutants. In: Proceedings of the National Academy of Sciences . Volume 66, Number 3, July 1970, pp. 773-779, PMID 4913210 , PMC 283117 (free full text).
  6. ^ Nelson SO, Wright JK & Postma PW The mechanism of inducer exclusion. Direct interaction between purified IIIGlc of the phosphoenolpyruvate: sugar phosphotransferase system and the lactose carrier of Escherichia coli. In: EMBO J. Vol. 2, pp. 715-720. PMC 555175 (free full text)
  7. ↑ Inada T., Kimata K. & H. Aiba (1996): Mechanism responsible for glucose-lactose diauxie in Escherichia coli: challenge to the cAMP model. In: Genes Cells Vol. 1, pp. 293-301. PMID 9133663
  8. ^ Anthony JF Griffiths, Jeffrey H. Miller, David T. Suzuki, Richard C. Lewontin, William M. Gelbart: Catabolite repression of the lac operon: positive control . 2000 ( nih.gov [accessed January 4, 2018]).
  9. ↑ F. Jacob & J. Monod (1961): Genetic regulatory mechanisms in the synthesis of proteins. In: J. Mol. Biol. Vol. 3, pp. 318-356. PMID 13718526
  10. ^ Information from the Nobel Foundation on the 1965 award ceremony to Francois Jacob and Jacques Monod (English)
  11. ↑ David P. Clark (2006): Molecular Biology, 1st ed. 2006, Elsevier GmbH, p. 248

See also

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