The lac operon (lactose operon) is the necessary operon for transport and lactose metabolism in Escherichia coli and many other enteric bacteria. Although glucose is the preferred carbon source for most bacteria, the lac operon allows for effective lactose digestion when glucose is not available through beta-galactosidase activity. The gene regulation of the lac operon is the first genetic regulatory mechanism to be clearly understood, thus becoming a prime example of prokaryotic gene regulation. This is often discussed in the molecular and cellular biology classes of the introduction for this reason. This lactose metabolism system is used by Jacob and Monod to determine how a cell knows which enzyme is synthesized. Their work on the lac operon won them the Nobel Prize in Physiology in 1965.
Bacterial operon is a polycistronic transcript capable of producing several proteins from one mRNA transcript. In this case, when lactose is needed as a source of sugar for bacteria, three lac operon genes can be expressed and their subsequent proteins are translated: lacZ , lacY and lacA . The lacZ gene product is? -actactosidase that cuts lactose, disaccharide, into glucose and galactose. lacY encodes the removal of Beta-galactoside, a protein that becomes embedded in the cytoplasmic membrane to allow the transport of lactose into the cell. Finally, lacA encode? -galactoside transacetylase.
It would be useless to produce enzymes when no lactose is available or if there is a preferred energy source, such as glucose. The lac operon uses a two-part control mechanism to ensure that the spent cells produce enzymes codonized by lac operon only when necessary. In the absence of lactose, repressor lac , lacI, discontinues the production of enzymes codonized by the lac operon . Lac replacement is always expressed unless the co-inducer ties it. In other words, it is only transcribed in the presence of a small molecule co-inducer. In the presence of glucose, the catabolite activator protein (CAP), required for enzyme production, remains inactive, and EIIA Glc closes the lactose intake to prevent the transport of lactose into the cell. This dual control mechanism causes successive utilization of glucose and lactose in two different growth phases, known as diauxie.
Video Lac operon
Structure
- Operon lac consists of three structural genes, and promoters, terminators, regulators, and operators. The three structural genes are: lacZ , lacY , and lacA .
- lacZ encode? -galactosidase (LacZ), an intracellular enzyme that cuts lactose disaccharides into glucose and galactose.
- lacY encodes Beta-galactoside permease (LacY), a transmembrane pumping sympasser? -galactosides include lactose into cells using a proton gradient in the same direction.
- lacA encode? -galactoside transacetylase (LacA), an enzyme that transfers acetyl groups from acetyl-CoA to? -galactosides.
Only lacZ and lacY are necessary for lactose catabolism.
Genetic Nomenclature
Three-letter abbreviations are used to describe phenotypes in bacteria including E. coli .
Examples include:
- Lac (ability to use lactose),
- His (ability to synthesize histidine amino acids)
- Mot (swimming motility)
- Sm R (resistance to streptomycin antibiotics)
In Lac's case, wild-type cells are Lac and are able to use lactose as a source of carbon and energy, while Lac - mutant derivatives can not use lactose. The same three letters are commonly used (lowercase, italicized) to label the genes involved in a particular phenotype, in which each different gene is also distinguished by the additional letters. The gene encoding enzyme lac is lacZ , lacY , and lacA . The fourth lac gene is lacI , encoding the lactose-repressor - "I" means inducibility .
One can differentiate between structural enzyme coding genes, and the protein encoding regulatory genes that affect gene expression. Current usage extends the phenotypic nomenclature to be applied to proteins: thus, LacZ is the protein product of the lacZ gene ,? -galactosidase. Short sequences that are not genes also affect gene expression, including lac promoter, lac p , and lac operator, lac o . Although not a strict use of standards, mutations that affect lac o are referred to as lac o c , due to historical reasons. Maps Lac operon
Rule
The specific control of the lac gene depends on the availability of lactose substrate to bacteria. Proteins are not produced by bacteria when lactose is not available as a carbon source. The lac genes are arranged into operons; that is, they are oriented in the same direction, which is directly adjacent to the chromosome and transcribed into a single polycistronic mRNA molecule. Transcription of all genes begins with the binding of an enzyme RNA polymerase (RNAP), a DNA binding protein, which binds to a specific DNA binding site, promoter, immediately upstream of the gene. The binding of RNA polymerase to the promoter is aided by a cAMP-bound catholic protein activator (CAP, also known as cAMP receptor protein). However, the lacI gene (the regulator gene for lac operus ) produces a protein that blocks RNAP from binding to the operon promoter. This protein can only be removed when allolactose binds it, and disables it. Proteins formed by the laci gene are known as lac repressors. The type of regulation that operon lac is referred to as negative induction, which means that the gene is off by a lacepressor factor (lactose) unless some molecule (lactose) has been added. Due to the presence of lac Repressor proteins, genetic engineers who replace the lacZ gene with other genes must grow experimental bacteria on agar with lactose available on it. Otherwise, the gene they are trying to express will not be expressed because the repressor protein still prevents RNAP from binding to the promoter and transcribing the gene. After the repressor is removed, RNAP then transcribes all three genes ( lacZYA ) to the mRNA. Each of the three genes on the mRNA strand has its own Shine-Dalgarno sequence, so the gene is independently decoded. The DNA sequence of E. coli lac operon, mRNA lacZYA , and the lacI genes are available from GenBank (view).
The first control mechanism is the regulatory response to lactose, which uses an intracellular regulatory protein called lactose-suppressor to inhibit production? -galactosidase in the absence of lactose. The lacI gene encodes the repressor near the operon lac and is always expressed ( constitutive ). If lactose is lost from the growth medium, the repressor binds very closely to the short DNA sequence right downstream of the promoter near the beginning of lacZ called the lac operator. The operator-binding repressor interferes with the binding of RNAP to the promoter, and therefore the LacZ and LacY mRNA encoding is only made at a very low level. When cells grow in the presence of lactose, however, a lactose metabolite called allolactose, made of lactose by the product of the lacZ gene, binds repressor, causing an allosteric shift. Thus changed, the repressor can not bind to the operator, allowing RNAP to transcribe the lac gene and thereby leads to a higher level of encoded protein.
The second control mechanism is the response to glucose, which uses a cytbolytic activator protein (CAP) homodimer to increase production? -galactosidase in the absence of glucose. Cyclic adenosine monophosphate (cAMP) is a signal molecule whose prevalence is inversely proportional to glucose. It binds to the CAP, which in turn allows the CAP to bind to the CAP binding site (DNA sequence 16 bp upstream of the promoter on the left in the diagram below, about 60 bp upstream of the transcription start site), which helps RNAP in binding to DNA. In the absence of glucose, high cAMP concentrations and binding of CAP-cAMP to DNA significantly increase the production of -galaktosidase, allowing cells to hydrolyze lactose and release galactose and glucose.
Recent inducer excretion is shown to block the expression of the operon lac when glucose is present. Glucose is transported into the cell by a phosphotransferase system that depends on PEP. The phosphoenolpyruvate phosphate group is transferred through a phosphorylated cascade comprised of public PTS proteos (phosphotransferases) of proteins HPr and EIA and glucose-specific PTS proteins EIIA Glc and EIIB Glc , the glucose transport cytoplasm of EII. The transport of glucose is accompanied by phosphorylation by EIIB Glc , depleting the phosphate group of other PTS proteins, including EIIA Glc . The unfrothofilated EIIA form Glc binds to the lac permease and prevents it from carrying lactose into the cell. Therefore, if glucose and lactose are present, glucose transport blocks the transport of the inducer from the lac operus.
Repressor structure
The lac substitute is a four-part protein, tetramer, with identical subunits. Each subunit contains a helix-turn-helix (HTH) motif capable of binding DNA. The operator site where the repressor represents is a series of DNA with repetitive inverted symmetry. The two parts of the DNA of the operator together bind two repressor subunits. Although two other reporeral subunits do nothing in this model, this property has not been understood for years.
It was discovered that two additional operators were involved in the lac regulation. One (O 3 ) lies about -90 bp upstream O 1 at the end of the lacI gene, and the other (O 2 ) is about 410 bp downstream O 1 at the beginning of lacZ . Both sites are not found at the beginning of work because they have excessive functions and individual mutations do not affect repression very much. Single mutations either to O 2 or O 3 have only 2 to 3 fold effects. However, their importance is indicated by the fact that double mutant defects in both O 2 and O 3 are dramatically not suppressed (about 70-fold).
In the current model, the lac repressor is tied simultaneously to the main operator O 1 and to O 2 or O 3 . DNA intervening rotates out of the complex. The redundant nature of two small operators shows that it is not an important specific circular complex. One idea is that the system works through withdrawal; if the bound repressor is released from O 1 for a while, binding to the minor operator keeps it around, so it can rebind quickly. This will increase the repressor affinity for O 1 .
Induction mechanism
Repressor is an allosteric protein, which can assume one of two slightly different forms, which are in equilibrium with each other. In one form of repressor it will bind the operator's DNA with high specificity, and in other forms it has lost its specificity. According to the classical model of induction, the binding of the inducer, either allolactose or IPTG, to the repressor affects the distribution of the repressor between the two forms. Thus, the repressor with the bound inducer is stabilized in a conformation that is not binding to DNA. However, this simple model can not be the whole story, since the repressor is bound enough stable to the DNA, but is released rapidly by the addition of the inducer. It is therefore apparent that the inducer may also bind the repressor when the repressor is attached to the DNA. It is not yet fully known what the actual binding mechanism is.
Non-specific binding role
The binding of non-specific repressors to DNA plays an important role in Lac-operon repression and induction. The specific binding sites for Lac-repressor proteins are operators. Non-specific interactions are mediated primarily by the charge-charge interactions while binding to operators is reinforced by hydrophobic interactions. In addition, there are many non-specific DNA sequences that can bind repressors. Basically, any sequence that is not an operator, is considered non-specific. Studies have shown that, in the absence of non-specific binding, induction (or disability) of the Lac-operon can not occur even with a saturated level of the inducer. It has been shown that, with no nonspecific bond, the basal induction rate is ten thousand times smaller than normally observed. This is because non-specific DNA acts as a kind of "sink" for repressor proteins, distracting them from the operator. Non-specific sequences decrease the number of available repressors in the cell. This in turn reduces the number of inducers needed to unrepress the system.
lactose analogy
A number of lactose or analog derivatives have been described which are useful for working with lac operon. These compounds are mainly substituted galactosides, in which the lactose glucose part is replaced by other chemical groups.
- Isopropil -? - D-thiogalactopyranoside (IPTG) is often used as an inducer of lac operus for physiological work. [1] IPTG binds to a repressor and disables it, but is not a substrate for? -galactosidase. One advantage of IPTG for in vivo studies is that it can not be metabolized by E. coli the concentration is fixed and the rate of controlled lacp/o controlled gene expression, is not a variable in the experiment. Intake of IPTG depends on the lactose permeate action in P. fluorescens , but not in E. coli .
- Phenyl -? - D-galactose (phenyl-Gal) is a substrate for? -galactosidase, but does not inactivate the repressor and therefore is not an inducer. Because wild-type cells produce very little? -galactosidase, they can not grow on phenyl-Gal as a source of carbon and energy. Mutants lacking repressors can grow on phenyl-Gal. Thus, a minimal medium containing only phenyl-Gal as a carbon and energy source is selective for mutant repressors and mutant carriers. If 10 8 cells of wild-type strains are coated on the agar plate containing phenyl-Gal, a rare colony that grows mainly is a spontaneous mutant affecting the repressor. The relative distributions of repressors and mutant operators are influenced by the target size. Since the coding repressor laci is about 50 times larger than the operator, mutant repressor dominates the election.
- Thiomethyl galactosidase [TMG] is another lactose analogue. This inhibits LACI repressor. At low inducer concentrations, both TMG and IPTG can enter cells via lactose permease. But at high inducer concentrations, the two analogs can enter the cell independently. TMG can reduce growth rates at high extracellular concentrations.
- Other compounds serve as colorful indicators of the -galactosidase activity.
- ONPG is split to produce very yellow compounds, orthonitrophenol and galactose, and is generally used as a substrate for testing? -galactosidase in vitro .
- The resulting colony? -galactosidase is blue by X-gal (5-bromo-4-chloro-3-indolyl-D-galactoside) which is an artificial substrate for B-galactosidase whose cleavage produces galactose and 4-Cl, 3-Br indigo resulting dark blue.
- Allolactose is a lactose isomer and is an inducer of lac operon. Lactose is galactose - (? 1- & gt; 4) - glukosa, while allolactose is galactose - (? 1- & gt; 6) -glukosa. Lactose is converted to allolactose by -galactosidase in an alternative reaction to the hydrolytic. The physiological experiment that shows the role of LacZ in the production of the "right" inducer in cell E. coli is the observation that the null mutant of lacZ can still produce lac bleach if grown with IPTG but not when grow with lactose. The explanation is that lactose processing into allolactose (catalyzed by -galaktosidase) is required to produce the inducer in the cell.
Classic model development
The experimental microorganisms used by FranÃÆ'§ois Jacob and Jacques Monod are common laboratory bacteria, E. coli , but many of the basic setting concepts found by Jacob and Monod are crucial for cellular regulation in all organisms. The main idea is that proteins are not synthesized when they are not needed - E. coli saves cellular and energy resources by not making three Lac proteins when it does not need to metabolize lactose, just like when other sugars like glucose are available. The following section discusses how E. coli controls certain genes in response to metabolic needs.
During World War II, Monod tested the effect of sugar combinations as a source of nutrition for E. coli and B subtilis. Monod followed up similar research that has been done by other scientists with bacteria and yeast. He found that bacteria that grow with two different sugars often display two growth phases. For example, if glucose and lactose are given, glucose is metabolized first (growth phase I, see Figure 2) and then lactose (growth phase II). Lactose is not metabolized during the first part of the diauxic growth curve because? -galactosidase is not made when glucose and lactose are present in the medium. Monod named this phenomenon diauxie.
Monod then focuses on the induction of formation -galaktosidase that occurs when lactose is a single sugar in the culture medium.
Mutual mutant classification
Jacob and Monod's conceptual breakthroughs are to recognize the difference between regulators and sites where they act to alter gene expression. A former soldier, Jacob uses the analogy of a bomber who will release his deadly charge after receiving a transmission or a special radio signal. The work system requires a soil transmitter and receiver on an airplane. Now, assume that the usual transmitter is damaged. This system can be made to work with the introduction of a second functional transmitter. Instead, he says, consider a bomber with a defective receiver. The bomber behavior is can not be changed with the introduction of a second functional aircraft.
To analyze the regenerative mutants of the lac operon, Jacob developed a system by which copies of both lac laci ( lacI genes with their promoters, and lacZYA with promoters and operators) can be entered into a single cell. Cultures of such bacteria, which are diploid for lac genes but otherwise normal, are then tested for the regeneration phenotype. Specifically, it is determined whether Lacz and LacY are made even in the absence of IPTG (since the lactose-suppressor produced by the mutant gene becomes non-functional). This experiment, in which a gene or a gene cluster is tested in pairs, is referred to as a complement test .
This test is illustrated in the image ( lacA omitted for simplicity). First, a particular haploid condition is displayed (ie the cell carries only one copy of the lac gene). Panel (a) denotes repression, (b) indicates induction by IPTG, and (c) and (d) shows the effect of mutation to gene lacI or to operator, respectively. In the panel (e) the complementation test for the repressor is displayed. If one copy of the lac gene carries the mutation in lacI , but the second copy is wild type for lacI , the resulting phenotype is normal- - but lacZ expressed when exposed to an IPTG inducer. Mutations affecting the repressor are said to be recessive for the wild type (and wild type is dominant ), and this is explained by the fact that the repressor is a small protein that can diffuse. in the cell. A copy of a lac operon adjacent to the damaged laci gene is effectively turned off by a protein produced from a second copy of lacI.
If the same experiment is performed using an operator mutation, different results are obtained (panel (f)). The cell phenotype that carries one mutant and one wild type operator site is that LacZ and LacY are produced even in the absence of an IPTG inducer; because the site operator is damaged, does not allow repressor binding to inhibit the transcription of structural genes. Dominant operator mutation. When the operator site where the repressor should be tied is damaged by mutation, the presence of a second functional site in the same cell makes no difference to the gene expression controlled by the mutant site.
A more sophisticated version of this experiment uses the tag tagged to distinguish between two copies of the lac gene and shows that unregulated structural genes are (one) one. (s) in addition to the mutant operator (panel (g). For example, suppose that one copy is marked by a mutation that disables lacZ so that it can produce only LacY proteins while the second copy carries a mutation affecting lacY and can only produce LacZ In this version, only a copy of the lac operand adjacent to the mutant operator declared without IPTG We say that the carrier mutation is cis-dominant , dominant for wild type but affects only a copy of the operon directly adjacent to it.
This explanation is misleading in an important sense, because of the result of the experimental description and then describes the results in terms of the model. But in fact, it is often true that the model comes first, and experiments are tailor-made to test the model. Jacob and Monod first imagine that there should be a site in DNA with operator properties, and then design a complementary test to show this.
The mutant operator dominance also shows the procedure for selecting them specifically. If the regulatory mutants are selected from wild type cultures using phenyl-Gal, as described above, operator mutations are rare compared to mutant repressors because the target size is very small. But if we start with a strain carrying two copies of the entire region of lac (diploid for lac ), a repressor mutation (which still happens) is not found. because complementation by the second type of gene, wild type lacI gives wild type phenotype. In contrast, a single copy of the operator confers mutant phenotype because it is dominant in the second wild-type copy.
Regulation by cyclic AMP
Diauxie's explanation depends on the characterization of additional mutations affecting the lac gene in addition to that described by the classical model. The other two genes, cya and crp , are then identified that are mapped away from lac , and that, when mutated, results in a decreasing rate of expression in presence IPTG and even in less repressive bacterial strains or carriers. The discovery of cAMP in E. coli led to a demonstration that mutants damaged by the cya genes but not crp genes could be returned to full activity with the addition of cAMP to the medium.
The cya gene encodes adenylate cyclase, which produces cAMP. In mutant cya , the absence of cAMP makes the lacZYA gene expression more than ten times lower than normal. The addition of cAMP corrects the low Lac expression characteristics of the mutant cya . The second gene, crp , encodes a protein called catabolite activating protein (CAP) or cAMP receptor protein (CRP).
However, the enzyme lactose metabolism is made in small amounts in the presence of glucose and lactose (sometimes called leaky expression) due to the fact that the LacI repressor rapidly associates from DNA and is not tightly bound to it, which can give RNAP time to bind and write mRNA from lacZYA . A leaky expression is required to allow the metabolism of some lactose after the glucose source is removed, but before the expression lac is fully activated.
Conclusion:
- When lactose is absent the Lac enzyme production is very small (the operator has Lac represses attached to it).
- When lactose is present but a preferred carbon source (such as glucose) also exists then a small amount of the enzyme is produced (Lac repressor is not attached to the operator).
- When glucose is absent, CAP-cAMP binds to the specific DNA site upstream of the promoter and makes the interactions of the proteins directly with RNAP that facilitate the binding of RNAP to the promoter.
The delay between growth phases reflects the time required to produce sufficient amounts of lactose-metabolic enzymes. First, CAP-regulating proteins must converge on the lac promoter, which results in increased production of lac \ i1 mRNA. More copies are available from the lac \ i> lac results in production (see translation) from more copies of Lacz (? -actactosidase, for lactose metabolism) and LacY (lactose allows to transport lactose into cells). After a delay is required to increase the enzyme levels of lactose metabolism, the bacteria enter into a rapid new cell growth phase.
Two catabolite suppression puzzles relate to how cAMP levels are coupled with the presence of glucose, and secondly, why cells should bother. After lactose is cleaved, it is actually a form of glucose and galactose (easily converted into glucose). In metabolic terms, lactose equals both carbon and energy sources as glucose. The cAMP level is not associated with intracellular glucose concentration but at the level of glucose transport, which affects the activity of adenylate cyclase. (In addition, glucose transport also leads to direct inhibition of the lactose permease.) As to why E. coli works in this way, one can only speculate. All bacteria ferment glucose, which indicates they often experience it. It is possible that small differences in transport efficiency or glucose metabolism v. Lactose makes it advantageous for the cell to regulate the operon lac in this way.
Use in molecular biology
The lac genes and their derivatives can be used as reporter genes in a number of bacteria-based techniques such as two hybrid analyzes, in which the successful binding of transcriptional activators to specific promoter sequences. must be determined. In the LB plate containing X-gal, the color change from the white to blue colonies corresponds to about 20-100 units of -galactosidase, while tetrazolium lactose and MacConkey lactose have a range of 100-1000 units, the most sensitive in the high and low sections of the range this one respectively. Since MacConkey lactose and tetrazolium lactose media depend on lactose-cleavage products, they require the presence of lacZ and lacY genes. Many lac fusion techniques include only the lacZ genes so they are suitable for X-gal dishes or ONPG liquid broth.
See also
- catabolit suppression
References
External links
- Lac Operon in the US National Library of Medicine Subject Medical Headings (MeSH)
- lac operon on NCBI Bookshelf [2]
- Virtual Cell Animation Collection Introducing: Lac Operon
- lac Operon: Science Bozeman
- Whole Mouse Whole Coloring for? -Galactosidase (lacZ) Activity
Source of the article : Wikipedia
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