Lac Operon
Prokaryotes (such as bacteria) regulate expression of their genes in different ways to eukaryotes. One method unique to prokaryotes is the use of operons. The most commonly referred to operon is the lac operon, and I have used (poorly drawn) diagrams in this post to try and make its function clear. The lac operon is described as an 'inducible' operon as it remains 'off' unless it is in the presence of an inducer.
This diagram is a very simplified version of what the DNA sequence that comprises the lac operon look like. Below is a
summary of what each part of the operon does.
LacI gene: While not actually part of the lac operon, it performs an important
regulatory function. The lacI gene codes for an mRNA that is then translated into a
repressor protein.
Promoter: The promoter region is a DNA sequence to which
RNA polymerase binds to begin transcription of the subsequent genes.
Operator: The operator region is a DNA sequence which allows
transcription factors to bind and either repress or enhance transcription of the subsequent genes. In this case, the transcription factor is the
repressor protein which inhibits expression of the lac genes.
LacZ gene: Encodes the enzyme
B-galactosidase (breaks down lactose into glucose and galactose)
LacY gene: Encodes the enzyme
lactose permease (pumps lactose into the cell)
LacA gene: Encodes the enzyme
B-galactoside transacetylase (transfers acetyl group from acetyl-Coenzyme A to B-galactosides - however its involvement in lactose catabolism is relatively unknown)
How the lac operon works
The important thing to remember about operons is that they are either constantly on or constantly off and rely on
positive or negative regulation by transcription factors
(repressors and enhancers) to switch them on or off. The lac operon is generally switched off unless
1) glucose levels are low AND ALSO
2) lactose levels are high in the prokaryote's surroundings. Both of these criteria must be met for the lac operon to be switched on and begin to convert lactose to glucose and galactose. This is because the lac operon
requires energy to hydrolyze (break down) lactose, so it is
more efficient to use glucose to meet the cell's energy demands, as it does not need to be broken down before use. Therefore, the operon will only function if there is no glucose to use as an energy source and if there is lactose available. If there is
both glucose and lactose in the cell's surroundings, the lac operon will not be switched on until all the glucose has been used first.
I have written the slightly
simpler version in
green, the
moderate difficulty version in
black and the slightly more
advanced content in
red so you can choose which information to read depending on the level of understanding you want to attain. The first part is the core details of the lac operon's function and focuses on the negative regulation of the lac operon through the binding of the repressor protein in the absence of an inducer. The second part is slightly more advanced and is about the positive regulation of the lac operon through the CAP-cAMP complex.
NOTE: Negative regulation involves the binding of a repressor protein to an operator to prevent transcription and
positive regulation involves the binding of a transcription factor to a promoter to enable transcription to occur.
General Details/Negative Regulation
- A molecule called a 'repressor' attaches to the 'operator' area of the operon. The operon is switched off. This prevents RNA polymerase from reading the lac genes so none of the lac enzymes are produced.
- When lactose is present, it binds to the repressor protein, causing it to detach from the operon so it is switched on.
- The genes can then be read and the lac enzymes produced
- These enzymes break down the lactose and also allow more lactose to enter the cell
- When there is no lactose left in the surroundings, the repressor reattaches to the operon again and the operon is switched off.
- The LacI gene codes for the repressor protein that binds to the operator region of the operon (though it also overlaps the promoter region). This repressor remains constantly bound unless the criteria mentioned above are fulfilled (low glucose, high lactose). The repressor acts by preventing RNA polymerase from binding to the promoter and blocking its path so it is unable to transcribe the Lac Z, Y and A genes.
- When an inducer is added (usually lactose, though artificial inducers such as IPTG can be added in labs), it binds to the repressor protein and causes a conformational change (an alteration in the shape of the protein), which causes the repressor to dissociate (release) from the operator region. This then allows the RNA polymerase to move along the DNA and transcribe (copy into a strand of mRNA) the lac genes.
- The lac genes work by producing B-galactosidase to break down the lactose into the two glucose and galactose for the cell to use, and lactose permease which increases the permeability of the cell to lactose so more lactose can enter.
- When the lactose in the surroundings reduces, or if glucose is added, the repressor rebinds and the operon is switched off so the lac enzymes stop being produced.
- The LacI gene codes for the repressor protein that binds to the operator region of the operon (though it also overlaps the promoter region). This repressor remains constantly bound unless the criteria mentioned above are fulfilled (low glucose, high lactose). The repressor acts by preventing RNA polymerase from binding to the promoter and blocking its path so it is unable to transcribe the Lac Z, Y and A genes.
- When an inducer is added, it binds to the repressor protein and causes a conformational change (an alteration in the shape of the protein), which causes the repressor to dissociate (release) from the operator region. The inducer is generally allolactose, which is an isomer of lactose and is produced as an intermediate substance as the lactose is metabolized. However, other artificial inducers such as IPTG can be used in labs to produce the same effect. This then allows the RNA polymerase to move along the DNA and transcribe (copy into a strand of mRNA) the lac genes.
- The lac genes work by producing lac enzymes. These enzymes are normally present in the bacteria at very low levels but increase rapidly in the presence of lactose. LacZ codes for B-galactosidase to hydrolyze the disaccharide lactose into the two monosaccharide sugars glucose and galactose for the cell to use, and LacY codes for lactose permease which increases the permeability of the cell to lactose so more lactose can enter. Lactose permease is a symporter, and transports B-galactosides such as lactose into the cell using a proton gradient in the same direction.
- When the lactose in the surroundings reduces, or if glucose is added, the repressor rebinds and the operon is switched off so the lac enzymes stop being produced.
Positive Regulation
Positive regulation serves to enhance the process described above and ensures that, even if lactose levels are high, the
lac operon won't be switched on unless glucose levels are low. As mentioned earlier, this is so the cell does not expend energy hydrolyzing lactose if there is still glucose available.
The way this works is through the formation of a CAP-cAMP complex.
This process is an additional regulatory mechanism to switch off the lac operon in the presence of glucose and is referred to as "catabolite repression".
cAMP = Cyclic adenosine
CAP = Catabolite Activator Protein - in the presence of cAMP,
When glucose is available to the cell in the surroundings, it inhibits (prevents) the function of an enzyme called adenylate cyclase. This enzyme is responsible for producing cyclic adenosine monophosphate (cAMP). Therefore, when glucose levels fall, cAMP levels will then rise. When cAMP levels are high, it forms a complex with a regulatory protein called the catabolite activator protein (CAP) and causes it to bind upstream of the lac promoter. This enhances binding of the RNA polymerase to the promoter, encouraging transcription of the operon and the use of lactose (as there is little glucose, lactose must be used instead). The lac operon would still be transcribed without the binding of CAP-cAMP but only at very low levels.
To summarise:
Glucose low ---> cAMP levels high (as enzyme that forms cAMP is no longer inhibited by the glucose) ---> CAP binds to site upstream of promoter --> RNA polymerase binds better --> Transcription of operon occurs at a high level --> Lactose used by cell
If you have any questions about anything covered in this post, please let me know in the comments and I will get back to you as soon as I can!
