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Gene regulation in bacteria - Regulation of the lac operon

Q: What does the lacI gene encode for?

Lac repressor, a protein that regulates lac operon by binding to the operator site.

57 important questions on Gene regulation in bacteria - Regulation of the lac operon

Which researchers investigated gene regulation in the beginning of the twentieth century, and gave us remarkable insights in this phenomenon?

Jacob and Monod.

What was the observation of Jacob and Monod of lactose metabolism in E. Coli?

They discovered that the increase of the activity of lactose-utilizing enzymes when exposed to lactose was due to the increased synthesis of the proteins that form the enzymes.

In bacteria, it is common for genes to be arranged in an operon. What is an operon?

A group of two or more genes that are under the transcriptional control of one promoter.

What does an operon encode?

A polycistronic mRNA, an RNA that consists of the sequences of more than one gene.

What is an advantage of an operon?

The genes often have a common functional goal, so the cell can coordinately regulate that group of genes.

What is the function of β-galactosidase?

- Cleaves lactose into galactose and glucose;
- Converts some of the lactose into allolactose (which functions as a small effector molecule for regulating lac operon).

What is lactose permease?

A protein required for the transport of lactose into the cytoplasm.

What is the function of galactoside transacetylase?

It attaches hydrophobic acetyl groups to lactose and lactose analogs. These molecules then can diffuse out of the cell. This prevents a toxic buildup.

What is the CAP site?

A DNA sequence recognized by an activator protein.

What is the operator site?

A DNA sequence that functions as a binding site for a repressor protein called lac repressor.

Besides the lac operon, in this part of the DNA is another transcriptional unit present. What is the other one called?

The lacI gene.

What does the lacI gene encode for?

Lac repressor, a protein that regulates lac operon by binding to the operator site.

In which 2 ways can the lac operon be transcriptionally regulated?

1. Inducible and under negative control;
2. Catabolite repression.

What does the first way of transcriptional regulation involve?

Lac repressor.

When is lac repressor bound to the operator site most of the time?

In the absence of allolactose.

How does allolactose influence the ability of the binding of lac repressor to the operator?

Lac repressor has four binding sites for allolactose. When four molecules of allolactose bind to the repressor, the structure of lac repressor is altered so that it is unable to bind to the operator.

When lac repressor is not able to bind to the operator site, what is the effect on the expression of the lac operon?

RNA polymerase is free to transcribe the operon.

How is it called when RNA polymerase is free to transcribe a gene?

The gene or operon has been induced.

How are the sites in an allosteric protein where small effector molecules bind to, called?

Allosteric sites.

In what do mutations in the lacI result?

In the constitutive expression of the lac operon (in presence & absence of lactose).

What are super-repressor mutations?

Another kind of lacI mutations that have the opposite effect: it results in a lac repressor protein that is unable to bind allolactose, and therefore lac repressor will remain bound to the operator site, so induction cannot occur.

What happens when a bacterial cell is exposed to lactose (steps)?

1. A small amount is transported into the cytoplasm via lactose permease;
2. Beta-galactosidase converts some of the lactose into allolactose;
3. Allolactose binds to lac repressor;
4. Lac repressor falls off the operator site;
5. Lac operon proteins are synthesized and lactose is metabolized.

What happens when all the lactose is depleted?

The allolactose levels decrease, and allolactose falls off the repressor. The repressor binds to the operator site again, and the expression of the lac operon is stopped.

What did Jacob and Monod initial think of the lacI mutation?

They did not know yet the function of the repressor, so they thought that the lacI mutation resulted in the synthesis of an activator, making it unnecessary for the cell to be exposed to lactose for the expression of the lac operon.

What are F factors?

Circular segments of DNA that are transferred during bacterial conjugation from one bacteria to another.

What is a merozygote?

A strain of bacteria containing F' factors, also called partial diploid.

What kind of F factors identified Jacob and Monod in their studies?

F' factors that carried the lacI gene and the lac operon.

What was the experimental approach of Jacob and Monod?

They used the production of merozygotes to determine the function of the lacI gene.

On which 2 key points was this experimental approach based?

- The two lacI genes in a merozygote may be different alleles (for example, one mutant and one normal);
- The genes on the bacterial chromosome and the genes on the F' factor are not adjacent to each other.

What do we now know of the second key point of Jacob and Monod?

That it is possible that the functional repressor proteins expressed from lacI on the F' factor can bind to the operator site of the lac operon located on the chromosome.

How was the experiment of Jacob and Monod set up (steps)?

- They let mutant strain and merozygote strain grow separately and divided each one into two tubes.
- They added lactose to one of the two tubes, incubated them and lysed them (to let beta-galactosidase escape from the cells);
- They added ONPG, which makes a yellow product in reaction with beta-galactosidase;
- They measured the yellow color with a spectrophotometer.

What are the results of Jacob and Monod's experiment?

- It did not matter whether or not lactose was added to the mutant strain, in those tubes beta-galactosidase was produced (constitutive expression);
- When no lactose was added to the merozygote, the level of beta-galactosidase was low. When lactose was added, it was high.

How can it be explained that in the merozygote strain even the bacterial chromosomal lac operon was expressed when lactose was added?

This result is consistent with the idea that lacI encodes for a repressor that can move around the cell.

What is a trans-effect?

A form of genetic regulation that can occur even though two DNA segments are not adjacent.

How is a regulatory protein, such as lac repressor, called?

A trans-acting factor.

How is a DNA segment that must be adjacent to the genes that it regulates, called?

A cis-acting element, which has a so-called cis-effect-

As discussed up to now, the lac operon is regulated by a repressor protein. By which kind of protein is the lac operon also regulated?

By an activator protein.

How is this second way of lac operon transcriptional regulation called?

Catabolite repression.

By the presence of which substance is catabolite repression influenced?

The presence of glucose.

What doe the E.coli cells do when they are exposed to both glucose and lactose?

They first use glucose, and catabolite repression prevents the use of lactose.

How is the sequential use of two sugars by a bacterium known?

Diauxic growth.

Which sugar is always metabolized preferentially in diauxic growth?

Glucose.

Glucose itself does not bind to a regulatory protein. What kind of molecule does it use?

A small effector molecule, cyclic-AMP.

What happens when a bacterium is exposed to both glucose and lactose?

- The concentration of cAMP in the cell decreases;
- CAP does not bind to the CAP site;
- Transcription of lac operon is low.

What happens in the absence of both lactose and glucose?

Transcription is very low, because the repressor is bound to the operator. Cyclic-AMP levels are high, so CAP is also bound to the DNA.

What happens when only lactose is present?

Allolactose and cAMP levels are high. Allolactose binds to the repressor and prevents it from binding to the DNA. Cyclic-AMP binds to CAP, and CAP binds to the CAP site. Transcription is high.

Why is this mechanism called catabolite repression?

Because in the presence of glucose (a catabolite) the transcription rate of lac operon is low (so it is repressed).

Why does this term seem inaccurate?

Because there is no repressor protein involved, only an inducer (cAMP) and an activator protein (CAP).

Is CAP only used in lac operon?

No, there are more than 100 operons in E.coli that can be activated by CAP.

In what way is the reality more complex than was originally thought?

The lac operon has 3 different operator sites instead of 1.

What is the order in which O1, 2, and 3 and the promoter (P) exist in the lac operon?

O3 - P - O1 - O2

To which of the 3 operons does the repressor bind?

Always to 2 operons, it binds to O3 and O1, or O1 and O2, but not at O2 and O3.

The missing of which operon does have the most effect on the level of repression?

The missing of O1.

What happens when O2 or O3 is missing?

It is less likely that the repressor will bind, so repression is reduced.

How can the repressor bind to the operator sites, even when they are not close to each other?

The DNA forms a loop to bring them close to each other.

What kind of molecule is lac repressor?

A tetramer, each dimer within the tetramer recognizes one operator site.

By what is the loop in the DNA formed that is needed for repressor to bind?

By cAMP-CAP complex, which causes a bend in the DNA structure.

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Genetics: Analysis & Principles II

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