Native to E. coli, the lac operon allows cells to use lactose (a sugar found in milk) for energy. Found only in prokaryotes, an operon is a contiguous row of genes transcribed from a single promoter. This means that genes within an operon are activated together and inactivated together. Most operons -- including the lac operon -- encode polycistronic mRNA, which is a continuous mRNA molecule encoding proteins involved in the same pathway. The anatomy of the lac operon is detailed below.
|I||Not part of the operon. Encodes a tetrameric repressor called lacI that binds to the operator (O) and blocks σ factor from binding to the promoter (P).|
|PlacZYA||A weak promoter for genes Z, Y and A that binds RNA Polymerase.|
|O||O (the operator) is where the repressor lacI binds. A mutation in the operator leads to constitutive expression, meaning constant high-level expression even without an inducer.|
|CBS||Where CRP binds, misleadingly titled CAP Binding Site (CBS).|
|Z||β-Galactosidase||β-galactosidase breaks lactose into glucose and galactose.|
|Y||Lac Permease||Lac permease transports lactose into the cell via an electrochemical gradient.|
|A||Thiogalactoside Transacetylase||Thiogalactoside transacetylase detoxifies the cell, since this process is toxic to the cell.|
The various factors involved in lactose metabolism were identified via direct analysis. Since these reactions are performed even in extracts lacking any cells, product formation was analyzed at every step of the way under various conditions. There are two regulatory forces: the repressor (lacI) and the activator (CRP). The repressor, lacI, binds to the operator and prevents transcription by blocking σ factor from binding to the promoter. The activator, CRP, is composed of cyclic AMP (cAMP) and CAP and binds to the CAP Binding Site. Once CRP binds to the CAP Binding Site, RNAP binds with high affinity to initiate transcription.
Glucose is easier for cells to catabolize (digest) than lactose, and the lac operon has evolved so the cell catabolizes only the best energy source available. Catabolization of glucose generates catabolites (byproducts) which lead to reduced cAMP levels. Without any cAMP to bind to CAP, no CRP can form and the lac operon is not activated. Thus, glucose concentrations are inversely proportional to cAMP concentrations -- high levels of glucose lead to low levels of cAMP, and vice versa.
|Β-Galactosidase||When inserted randomly into the mouse genome without associated control sequences, -galactosidase is not expressed in any tissue. However, when ligated to a 1kb sequence conserved in fish, Ligate and inject into fertilized mouse egg to generate a transgenic mouse with this DNA inserted randomly into some region of the genome. (Not gene targeting by homologous recombination.) Noncoding regions near a gene that is highly conserved across species (determined via comparative analysis) often a site for transcriptional control.|
|Allolactose||Allolactose is a byproduct of lactose metabolism that binds lacI (the repressor) to keep it form binding to the operator.|
|cAMP Receptor Protein||cAMP Receptor Protein (CRP) is a dimeric activator protein composed of cyclic AMP (cAMP) and CAP. CRP binds binds next to the promoter and helps RNA Polymerase bind the promoter, thus increasing transcription of lac and other operons.|
|Adenylate Cyclase||Adenylate Cyclase (cya) produces cAMP form ATP, and is inhibited when glucose is present.|
|Glucose||Glucose activates the phosphotransferase system (PTS). One component of the PTS -- IIa -- inhibits production of cAMP from ATP, thus lowering cAMP concentrations and preventing CRP (the activator) from forming.|
|Lactose||Lactose is transported into the cell by lac permease, then induces the lac operon to produce β-galactosidase. β-galactosidase breaks lactose down into glucose and galactose.|
|ONPG||Ortho-Nitrophenyl-β-galactoside is a colorless chemical cleaved by β-galactosidase to form galactose and orthonitrophenol (a yellow compound). This color formation allows for assay of β-galactosidase activity; however, ONPG does not induce the lac operon.|
|IPTG||Isopropyl β-D-1-thiogalactopyranoside mimics allolactose, a byproduct of lactose metabolism which induces the lac operon. IPTG does not require lac permease to enter the cell, and CRP must also be present to activate transcription.|
|cAMP||cyclic AMP (cAMP) is necessary for CRP to form and bind CBS; in absence of cAMP (even without repression) transcription is not activated. cAMP alone is not enough to activate transcription; the repressor has to be deactivated, or the operator (where the repressor binds) must be mutated.|
|Maltose||Not used preferably.|
|Mannitol||Mannitol is preferable over lactose; glucose is more preferable than mannitol.|