What is a lac operon?
The lac operon (lactose operon) is an operon required for the transport and metabolism of lactose in Escherichia coli and many other enteric bacteria.
- Escherichia coli and most other bacteria are capable of growth using any one of several carbohydrates (example: glucose, sucrose, galactose, arabinose, and lactose) as an energy source.
- If glucose is present in the environment, it will be preferentially metabolized by E.coli cells. However, in the absence of glucose, E. coli cells can grow very well on other carbohydrates.
- Lactose (a disaccharide) is one of the major carbohydrates found in milk which can be metabolized by E.coli bacteria that reside in the gut of mammals.
- Cells growing in medium containing the sugar lactose as the sole carbon source synthesize two enzymes, β -galactosidase and β-galactoside permease, which are uniquely required for the catabolism of lactose.
- Lactose does not easily diffuse across the E.coli cell membrane and must be actively transported into the cell by the enzyme permease.
- To utilize lactose as an energy source, E.coli must first break it into glucose and galactose, a reaction catalyzed by the enzyme β-galactosidase.
- The genes encoding these enzymes are located on lac operon.
- Neither of these enzymes is of any use to E.coli cells if no lactose is available to them.
- The synthesis of these two enzymes requires considerable energy (in the form of ATP and GTP). Thus, E.coli cells have evolved a regulatory mechanism by which the synthesis of these lactose-catabolizing enzymes is turned on in the presence of lactose and turned off in its absence.
(That means Lac operon is an inducible operon which is induced by its substrate lactose)
Structure of lac operon
The lactose or lac operon of Escherichia coli is a cluster of three structural genes encoding proteins involved in lactose metabolism and the sites on the DNA involved in regulation of the operon.
Structural genes in Lac operon
All the three structural genes are transcribed under the control of a common promoter into a polycistronic mRNA (contains multiple independent translation start (AUG) and stop (UAA) codons for each cistron) that get translated into individual enzymes.
1) lacZ: Encodes β-galactosidase enzyme (LacZ)
Two physiologically important reactions are catalysed by β – galactosidase
1) Conversion of lactose to allolactose (lac operon inducer) by transglycosylation.
2) Cleavage of disaccharide lactose to produce the monosaccharide glucose and galactose
(β – galactosidase cleaves 1, 4 linkage of lactose for releasing monosaccharide units)
2) lacY: Encodes β-galactoside permease enzyme (LacY)
It is a membrane-bound transport protein that actively transport lactose into the cell
3) lacA: Encodes β-galactoside transacetylase enzyme(LacA)
These enzymes are also known as galactoside O-acetyltransferase
Function: Protect the cell from the buildup of toxic products created by β-galactosidase acting on other galactosides. These enzymes appear to modify toxic galactosides to facilitate their removal from the cell.
How they act?
This enzyme transfers an acetyl group from acetyl-CoA to β-galactosides. By acetylating galactosides other than lactose, the transferase prevents β-galactosidase from cleaving them.
Only lacZ and lacY appear to be necessary for lactose catabolism.
Regulators in lac operon (cis and trans regulators)
In addition to the three protein-coding genes, the lac operon contains short DNA sequences that do not encode proteins, but are instead binding sites for proteins involved in transcriptional regulation of the operon.
They are: P (promoter), O (operator) and CBS (CAP-binding site).
These sequence elements are called cis-elements because they must be located on the same piece of DNA as the genes they regulate.
1) Lac promoter (lacP)
- The promoter is the DNA sequence of the operon recognized by DNA-dependent RNA polymerase.
- lac promoter is located immediately upstream of the structural genes. RNA polymerase binds to the promoter and moves down the DNA molecule, transcribing the structural genes.
- This promoter is common for all the three structural genes.
2) Operator (lacO)
- It is the binding site for the regulatory protein called repressor.
3) CAP –binding site
- The CAP binding site is a positive regulatory site that is bound by catabolite activator protein (CAP).
- CAP-binding site is located upstream of the RNA polymerase binding site in the promoter.
- This increases the binding ability of RNA polymerase to the promoter region and the transcription of the genes.
trans regulators: The proteins that bind to these cis-elements are called trans-regulators because (as diffusible molecules) they do not necessarily need to be encoded on the same piece of DNA as the genes they regulate.
Example: Lac repressor, CAP
1) Lac repressor – LacI (allosterically regulated repressor)
Structure: Tetramer with identical subunits (homotetramer)
It has two binding sites (one for Allolactose binding and one for DNA binding)
Each subunit contains a helix-turn-helix (HTH) motif capable of binding to DNA.
Gene encoding: lacI gene (Located upstream of the lac promoter and has its own promoter (PI). The lacI gene is transcribed into a short mRNA that is translated into a repressor
Binding site on DNA: Binds to operator sequences adjacent to the promoter of the lac operon
(In the absence of lactose the repressor binds to the lac operator site lac O)
Effect of repressor binding to operator: Binding of the repressor prevents RNA polymerase from binding to the promoter. Therefore, the operon will not be transcribed when the operator is occupied by a repressor.
2) CAP (cAMP binding protein)
- CAP is an allosteric activator of the lac operon
- Binds to specific cis-element within the lac promoter called the CAP binding sequence (CBS).
- When CAP is bound to at CBS, RNA polymerase is better able to bind to the promoter and initiate transcription.
The lac operon is inducible: Regulator gene produces a repressor that binds to the operator site and prevents the transcription of the structural genes. The presence of allolactose inactivates the repressor and allows the transcription of the lac operon.
Point to remember
A gene is generally italicized in lower case and the encoded protein, when abbreviated, is expressed in roman type with the first letter capitalized.
For example, the gene lacI encodes the repressor protein LacI.