Lac operon


The lactose operon is an operon required for the transport and metabolism of lactose in E. coli and many other enteric bacteria. Although glucose is the preferred carbon source for most enteric bacteria, the lac operon allows for the effective digestion of lactose when glucose is not available through the activity of β-galactosidase. Gene regulation of the lac operon was the first genetic regulatory mechanism to be understood clearly, so it has become a foremost example of prokaryotic gene regulation. It is often discussed in introductory molecular and cellular biology classes for this reason. This lactose metabolism system was used by François Jacob and Jacques Monod to determine how a biological cell knows which enzyme to synthesize. Their work on the lac operon won them the Nobel Prize in Physiology in 1965.
Most bacterial cells including E. coli lack introns in their genome. They also lack a nuclear membrane. Hence the gene regulation by lac operon occurs at the transcriptional level, by controlling transcription of DNA.
Bacterial operons are polycistronic transcripts that are able to produce multiple proteins from one mRNA transcript. In this case, when lactose is required as a sugar source for the bacterium, the three genes of the lac operon can be transcribed and their subsequent proteins translated: lacZ, lacY, and lacA. The gene product of lacZ is β-galactosidase which cleaves lactose, a disaccharide, into glucose and galactose. lacY encodes β-galactoside permease, a membrane protein which becomes embedded in the Plasma membrane to enable the cellular transport of lactose into the cell. Finally, lacA encodes β-galactoside transacetylase.
Layout of the lac operon.
Note that the number of base pairs in diagram given above are not to scale. There are in fact over 5300 base pairs in the lac operon.
It would be wasteful to produce enzymes when no lactose is available or if a preferable energy source such as glucose were available. The lac operon uses a two-part control mechanism to ensure that the cell expends energy producing the enzymes encoded by the lac operon only when necessary.
In the absence of lactose, the lac repressor, encoded by lacI, halts production of the enzymes and transport proteins encoded by the lac operon. It does so by blocking the DNA dependent RNA polymerase. This blocking/ halting is not perfect, and a minimal amount of gene expression does take place all the time. The repressor protein is always expressed, but the lac operon are almost completely repressed, allowing for a small level of background expression. If this weren't the case, there would be no lacY transporter protein in the cellular membrane; consequently, the lac operon would not be able to detect the presence of lactose.
When lactose is available but not glucose, then some lactose enters the cell using pre-existing transport protein encoded by lacY. This lactose then combines with the repressor and inactivates it, hence allowing the lac operon to be expressed. Then more β-galactoside permease is synthesized allowing even more lactose to enter and the enzymes encoded by lacZ and lacA can digest it.
However, in the presence of glucose, regardless of the presence of lactose, the operon will be repressed. This is because the catabolite activator protein, required for production of the enzymes, remains inactive, and EIIAGlc shuts down lactose permease to prevent transport of lactose into the cell. This dual control mechanism causes the sequential utilization of glucose and lactose in two distinct growth phases, known as diauxie.

Structure

  • The lac operon consists of 3 structural genes, and a promoter, a terminator, regulator, and an operator. The three structural genes are: lacZ, lacY, and lacA.
  • *lacZ encodes β-galactosidase, an intracellular enzyme that cleaves the disaccharide lactose into glucose and galactose.
  • * lacY encodes β-galactoside permease, a transmembrane symporter that pumps β-galactosides including lactose into the cell using a proton gradient in the same direction. Permease increases the permeability of the cell to β-galactosides.
  • * lacA encodes β-galactoside transacetylase, an enzyme that transfers an acetyl group from acetyl-CoA to thiogalactoside.
Only lacZ and lacY appear to be necessary for lactose catabolic pathway.
By numbers, lacI has 1100 bps, lacZ has 3000 bps, lacY has 800 bps, lacA has 800 bps, with 3 bps corresponding to 1 amino acid.

Genetic nomenclature

Three-letter abbreviations are used to describe phenotypes in bacteria including E. coli.
Examples include:
  • Lac,
  • His
  • Mot
  • SmR
In the case of Lac, wild type cells are Lac+ and are able to use lactose as a carbon and energy source, while Lac− mutant derivatives cannot use lactose. The same three letters are typically used to label the genes involved in a particular phenotype, where each different gene is additionally distinguished by an extra letter. The lac genes encoding enzymes are lacZ, lacY, and lacA. The fourth lac gene is lacI, encoding the lactose repressor—"I" stands for inducibility.
One may distinguish between structural genes encoding enzymes, and regulatory genes encoding proteins that affect gene expression. Current usage expands the phenotypic nomenclature to apply to proteins: thus, LacZ is the protein product of the lacZ gene, β-galactosidase. Various short sequences that are not genes also affect gene expression, including the lac promoter, lac p, and the lac operator, lac o. Although it is not strictly standard usage, mutations affecting lac o are referred to as lac oc, for historical reasons.

Regulation

Specific control of the lac genes depends on the availability of the substrate lactose to the bacterium. The proteins are not produced by the bacterium when lactose is unavailable as a carbon source.
The lac genes are organized into an operon; that is, they are oriented in the same direction immediately adjacent on the chromosome and are co-transcribed into a single polycistronic mRNA molecule. Transcription of all genes starts with the binding of the enzyme RNA polymerase, a DNA-binding protein, which binds to a specific DNA binding site, the promoter, immediately upstream of the genes. Binding of RNA polymerase to the promoter is aided by the cAMP-bound catabolite activator protein. However, the lacI gene produces a protein that blocks RNAP from binding to the operator of the operon. This protein can only be removed when allolactose binds to it, and inactivates it. The protein that is formed by the lacI gene is known as the lac repressor. The type of regulation that the lac operon undergoes is referred to as negative inducible, meaning that the gene is turned off by the regulatory factor unless some molecule is added. Once the repressor is removed, RNAP then proceeds to transcribe all three genes into mRNA. Each of the three genes on the mRNA strand has its own Shine-Dalgarno sequence, so the genes are independently translated. The DNA sequence of the E. coli ''lac operon, the lacZYA mRNA, and the lacI genes are available from GenBank .
The first control mechanism is the regulatory response to lactose, which uses an intracellular regulatory protein called the lactose repressor to hinder production of β-galactosidase in the absence of lactose. The lacI gene coding for the repressor lies nearby the lac operon and is always expressed. If lactose is missing from the growth medium, the repressor binds very tightly to a short DNA sequence just downstream of the promoter near the beginning of lacZ called the lac operator. The repressor binding to the operator interferes with binding of RNAP to the promoter, and therefore mRNA encoding LacZ and LacY is only made at very low levels. When cells are grown in the presence of lactose, however, a lactose metabolite called allolactose, made from lactose by the product of the lacZ gene, binds to the repressor, causing an allosteric shift. Thus altered, the repressor is unable to bind to the operator, allowing RNAP to transcribe the lac genes and thereby leading to higher levels of the encoded proteins.
The second control mechanism is a response to glucose, which uses the catabolite activator protein homodimer to greatly increase production of β-galactosidase in the absence of glucose. Cyclic adenosine monophosphate is a signal molecule whose prevalence is inversely proportional to that of glucose. It binds to the CAP, which in turn allows the CAP to bind to the CAP binding site, which assists the RNAP in binding to the DNA. In the absence of glucose, the cAMP concentration is high and binding of CAP-cAMP to the DNA significantly increases the production of β-galactosidase, enabling the cell to hydrolyse lactose and release galactose and glucose.
More recently inducer exclusion was shown to block expression of the lac operon when glucose is present. Glucose is transported into the cell by the PEP-dependent phosphotransferase system. The phosphate group of phosphoenolpyruvate is transferred via a phosphorylation cascade consisting of the general PTS proteins HPr and EIA and the glucose-specific PTS proteins EIIAGlc and EIIBGlc, the cytoplasmic domain of the EII glucose transporter. Transport of glucose is accompanied by its phosphorylation by EIIBGlc, draining the phosphate group from the other PTS proteins, including EIIAGlc. The unphosphorylated form of EIIAGlc binds to the lac permease and prevents it from bringing lactose into the cell. Therefore, if both glucose and lactose are present, the transport of glucose blocks the transport of the inducer of the lac'' operon.