Stefan Oehler

The three operators of the lac operon cooperate in repression

We tested the effect of systematic destruction of all three lac operators of the chromosomal lac operon of Escherichia coli on repression by Lac repressor. Absence of just one ‘pseudo‐operator’ O2 or O3 decreases repression by wild‐type tetrameric Lac repressor approximately 2‐ to 3‐fold; absence of both ‘pseudo‐operators’ decreases repression greater than 50‐fold. O1 alone represses under these conditions only approximately 20‐fold. Dimeric active Lac repressor (iadi) represses the wild‐type lac operon to about the same low extent. This indicates that cooperative interaction between lac operators is due to DNA loop formation mediated by tetrameric Lac repressor. Under conditions where loop formation is impossible, occupation of O3 but not of O2 may lead to weak repression. This suggests that under these conditions CAP activation may be inhibited and that stopping transcription at O2 does not significantly contribute to repression.

Induction of the lac promoter in the absence of DNA loops and the stoichiometry of induction

In vivo induction of the Escherichia coli lactose operon as a function of inducer concentration generates a sigmoidal curve, indicating a non-linear response. Suggested explanations for this dependence include a 2:1 inducer–repressor stoichiometry of induction, which is the currently accepted view. It is, however, known for decades that, in vitro, operator binding as a function of inducer concentration is not sigmoidal. This discrepancy between in vivo and in vitro data has so far not been resolved. We demonstrate that the in vivo non-linearity of induction is due to cooperative repression of the wild-type lac operon through DNA loop formation. In the absence of DNA loops, in vivo induction curves are hyperbolic. In the light of this result, we re-address the question of functional molecular inducer–repressor stoichiometry in induction of the lac operon.

Feedback Regulation of Lac Repressor Expression in Escherichia coli

Negative feedback regulation, mediated through repressor binding site O3, which overlaps the lacI gene, could explain the robustness of the weak expression of Lac repressor. Significant autorepression of Lac repressor has never been ruled out. In the work presented here, the degree of autoregulation of Lac repressor was determined. It is negligible.

Prokaryotic control of transcription: How and why does it differ from eukaryotic control?

Escherichia coli and all other prokaryotes have developed elaborate mechanisms to adapt their metabolism to a rapidly changing environment. Some of these mechanisms allow the bacteria to approach or to flee particular chemicals (Adler, 1975; Boyd and Simon, 1982). We will not discuss such mechanisms here. Other mechanisms adapt the transcription rates of genes whose products are needed or not needed in a particular environment. Genes which deal with the catabolism of chemicals which suddenly appear in the environment have to be rapidly turned on. We have to recall that the inner bacterial membrane does not allow the entry of most organic chemicals. There has to be a permease, a specific pump, present which transports the chemical into the cell.

Effector Overlap between the lac and mel Operons of Escherichia coli: Induction of the mel Operon with β-Galactosides.

The lac (lactose) operon (which processes β-galactosides) and the mel (melibiose) operon (which processes α-galactosides) of Escherichia coli have a close historical connection. A number of shared substrates and effectors of the permeases and regulatory proteins have been reported over the years. Until now, β-thiogalactosides like TMG (methyl-β-d-thiogalactopyranoside) and IPTG (isopropyl-β-d-thiogalactopyranoside) have not generally been considered to be inducers of the mel operon. The same is true for β-galactosides such as lactose [β-d-galactopyranosyl-(1→4)-d-glucose], which is a substrate but is not itself an inducer of the lac operon. This report shows that all three sugars can induce the mel operon significantly when they are accumulated in the cell by Lac permease. Strong induction by β-thiogalactosides is observed in the presence of Lac permease, and strong induction by lactose (more than 200-fold) is observed in the absence of β-galactosidase. This finding calls for reevaluation of TMG uptake experiments as assays for Lac permease that were performed with mel+ strains.IMPORTANCE The typical textbook picture of bacterial operons is that of stand-alone units of genetic information that perform, in a regulated manner, well-defined cellular functions. Less attention is given to the extensive interactions that can be found between operons. Well-described examples of such interactions are the effector molecules shared by the lac and mel operons. Here, we show that this set has to be extended to include β-galactosides, which have been, until now, considered not to effect the expression of the mel operon. That they can be inducers of the mel operon as well as the lac operon has not been noted in decades of research because of the Escherichia coli genetic background used in previous studies.

Induction of Transcription

There are two main strategies for the induction of transcription: a strong promoter is negatively controlled by a repressor protein and induction of transcription is effected by an inducer (a ligand that inactivates the repressor). Alternatively, a weak promoter is positively controlled by an activator protein bound to a coactivator (a ligand required for transcription activation). The lac operon, one of the paradigms of transcription regulation, is subject to both negative and positive control. A closer look at this system reveals that, while the principle of transcription induction seems simple, actual inducible biological systems tend to be complex.

“Cold-Sensitive” Mutants of the Lac Repressor

Thirteen of more than 4,000 single-amino-acid-replacement mutants of the Lac repressor, generated by suppression of amber nonsense mutants, were characterized as having a cold-sensitive phenotype. However, when expressed as missense mutations, none of the replacements cause cold sensitivity, implicating the suppression mechanism as being responsible for this phenotype.