File Name: positive and negative regulation of lac operon .zip
The lactose operon lac operon is an operon required for the transport and metabolism of lactose in E. Although glucose is the preferred carbon source for most bacteria, the lac operon allows for the effective digestion of lactose when glucose is not available through the activity of beta-galactosidase.
It is often discussed in introductory molecular and cellular biology classes for this reason. Their work on the lac operon won them the Nobel Prize in Physiology in 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 expressed and their subsequent proteins translated: lacZ , lacY , and lacA.
Finally, lacA encodes Galactoside acetyltransferase. 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 other words, it is transcribed only in the presence of small molecule co-inducer. In the presence of glucose, the catabolite activator protein CAP , required for production of the enzymes, remains inactive, and EIIA Glc 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.
Only lacZ and lacY appear to be necessary for lactose catabolism. The same three letters are typically used lower-case, italicized 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. 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 o c , for historical reasons. 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. 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 lac repressor unless some molecule lactose is added. Because of the presence of the lac repressor protein, genetic engineers who replace the lacZ gene with another gene will have to grow the experimental bacteria on agar with lactose available on it.
If they do not, the gene they are trying to express will not be expressed as the repressor protein is still blocking RNAP from binding to the promoter and transcribing the gene. Each of the three genes on the mRNA strand has its own Shine-Dalgarno sequence , so the genes are independently translated. The lacI gene coding for the repressor lies nearby the lac operon and is always expressed constitutive.
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. 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. Cyclic adenosine monophosphate cAMP is a signal molecule whose prevalence is inversely proportional to that of 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 unphosphorylated form of EIIA Glc 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. The lac repressor is a four-part protein, a tetramer, with identical subunits. The operator site where repressor binds is a DNA sequence with inverted repeat symmetry.
The two DNA half-sites of the operator together bind to two of the subunits of the repressor. Although the other two subunits of repressor are not doing anything in this model, this property was not understood for many years.
Eventually it was discovered that two additional operators are involved in lac regulation. These two sites were not found in the early work because they have redundant functions and individual mutations do not affect repression very much. Single mutations to either O 2 or O 3 have only 2 to 3-fold effects. However, their importance is demonstrated by the fact that a double mutant defective in both O 2 and O 3 is dramatically de-repressed by about fold.
In the current model, lac repressor is bound simultaneously to both the main operator O 1 and to either O 2 or O 3. The intervening DNA loops out from the complex. The redundant nature of the two minor operators suggests that it is not a specific looped complex that is important. One idea is that the system works through tethering; if bound repressor releases from O 1 momentarily, binding to a minor operator keeps it in the vicinity, so that it may rebind quickly.
This would increase the affinity of repressor for O 1. The repressor is an allosteric protein , i. In one form the repressor will bind to the operator DNA with high specificity, and in the other form it has lost its specificity. According to the classical model of induction, binding of the inducer, either allolactose or IPTG, to the repressor affects the distribution of repressor between the two shapes.
Thus, repressor with inducer bound is stabilized in the non-DNA-binding conformation. However, this simple model cannot be the whole story, because repressor is bound quite stably to DNA, yet it is released rapidly by addition of inducer. Therefore, it seems clear that an inducer can also bind to the repressor when the repressor is already bound to DNA.
It is still not entirely known what the exact mechanism of binding is. Non-specific binding of the repressor to DNA plays a crucial role in the repression and induction of the Lac-operon. The specific binding site for the Lac-repressor protein is the operator. The non-specific interaction is mediated mainly by charge-charge interactions while binding to the operator is reinforced by hydrophobic interactions.
Additionally, there is an abundance of non-specific DNA sequences to which the repressor can bind. Essentially, any sequence that is not the operator, is considered non-specific.
Studies have shown, that without the presence of non-specific binding, induction or unrepression of the Lac-operon could not occur even with saturated levels of inducer. It had been demonstrated that, without non-specific binding, the basal level of induction is ten thousand times smaller than observed normally. This is because the non-specific DNA acts as sort of a "sink" for the repressor proteins, distracting them from the operator. The non-specific sequences decrease the amount of available repressor in the cell.
This in turn reduces the amount of inducer required to unrepress the system. A number of lactose derivatives or analogs have been described that are useful for work with the lac operon. These compounds are mainly substituted galactosides, where the glucose moiety of lactose is replaced by another chemical group. The following section discusses how E. Monod was following up on similar studies that had been conducted by other scientists with bacteria and yeast.
He found that bacteria grown with two different sugars often displayed two phases of growth. For example, if glucose and lactose were both provided, glucose was metabolized first growth phase I, see Figure 2 and then lactose growth phase II. Monod named this phenomenon diauxie. A conceptual breakthrough of Jacob and Monod  was to recognize the distinction between regulatory substances and sites where they act to change gene expression.
A former soldier, Jacob used the analogy of a bomber that would release its lethal cargo upon receipt of a special radio transmission or signal. A working system requires both a ground transmitter and a receiver in the airplane. Now, suppose that the usual transmitter is broken. This system can be made to work by introduction of a second, functional transmitter.
In contrast, he said, consider a bomber with a defective receiver. The behavior of this bomber cannot be changed by introduction of a second, functional aeroplane. To analyze regulatory mutants of the lac operon, Jacob developed a system by which a second copy of the lac genes lacI with its promoter, and lacZYA with promoter and operator could be introduced into a single cell.
A culture of such bacteria, which are diploid for the lac genes but otherwise normal, is then tested for the regulatory phenotype. In particular, it is determined whether LacZ and LacY are made even in the absence of IPTG due to the lactose repressor produced by the mutant gene being non-functional. This experiment, in which genes or gene clusters are tested pairwise, is called a complementation test.
This test is illustrated in the figure lacA is omitted for simplicity. First, certain haploid states are shown i. Panel a shows repression, b shows induction by IPTG, and c and d show the effect of a mutation to the lacI gene or to the operator, respectively. In panel e the complementation test for repressor is shown. If one copy of the lac genes carries a mutation in lacI , but the second copy is wild type for lacI , the resulting phenotype is normal—but lacZ is expressed when exposed to inducer IPTG.
Mutations affecting repressor are said to be recessive to wild type and that wild type is dominant , and this is explained by the fact that repressor is a small protein which can diffuse in the cell. The copy of the lac operon adjacent to the defective lacI gene is effectively shut off by protein produced from the second copy of lacI. If the same experiment is carried out using an operator mutation, a different result is obtained panel f.
The phenotype of a cell carrying one mutant and one wild type operator site is that LacZ and LacY are produced even in the absence of the inducer IPTG; because the damaged operator site, does not permit binding of the repressor to inhibit transcription of the structural genes. The operator mutation is dominant. When the operator site where repressor must bind is damaged by mutation, the presence of a second functional site in the same cell makes no difference to expression of genes controlled by the mutant site.
A more sophisticated version of this experiment uses marked operons to distinguish between the two copies of the lac genes and show that the unregulated structural gene s is are the one s next to the mutant operator panel g. For example, suppose that one copy is marked by a mutation inactivating lacZ so that it can only produce the LacY protein, while the second copy carries a mutation affecting lacY and can only produce LacZ.
In this version, only the copy of the lac operon that is adjacent to the mutant operator is expressed without IPTG.
15: Positive and negative control of gene expression
An operon is a cluster of coordinately regulated genes. It includes structural genes generally encoding enzymes , regulatory genes encoding, e. The type of control is defined by the response of the operon when no regulatory protein is present. In the case of negative control , the genes in the operon are expressed unless they are switched off by a repressor protein. Thus the operon will be turned on constitutively the genes will be expressed when the repressor in inactivated. In the case of positive control , the genes are expressed only when an active regulator protein, e. Thus the operon will be turned off when the positive regulatory protein is absent or inactivated.
NCBI Bookshelf. An Introduction to Genetic Analysis. New York: W. Freeman; The inducer —repressor control of the lac operon is an example of negative control , in which expression is normally blocked. Figure distinguishes between these two basic types of control systems.
Regulation of Gene Expression. Cellular function is influenced by cellular environment. Adaptation to specific environments is achieved by regulating the expression of genes that encode the enzymes and proteins needed for survival in a particular environment. Factors that influence gene expression include nutrients, temperature, light, toxins, metals, chemicals, and signals from other cells. Malfunctions in the regulation of gene expression can cause various human disorders and diseases.
November 6, ]. The biological activities of each cell are largely determined by the active proteins expressed in it. But in cells, some proteins are found in very low quantities, and others are quite abundant, so how is this protein expression controlled? A cell needs a different enzyme to transport each molecule into the cell, and yet another to breakdown or change each of these molecules into something usable to the cell. If the cell simultaneously and constantly produced every enzyme it could possibly need, it would take more energy to produce all these enzymes than what would be derived from breaking down the molecules.
Operon model. An operon is a group of structural genes whose expression is coordinated by an operator. The repressor encoded by a regulatory gene binds to the operator and represses the transcription of operon. In the presence of inducer, the repressor is inactivated and dissociates from operator to express the operon. Thus, the expression of the operon is controlled by a cis -acting operator and by a trans -acting repressor.
The lactose operon lac operon is an operon required for the transport and metabolism of lactose in E. Although glucose is the preferred carbon source for most bacteria, the lac operon allows for the effective digestion of lactose when glucose is not available through the activity of beta-galactosidase. It is often discussed in introductory molecular and cellular biology classes for this reason.
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ГЛАВА 75 Пальцы Стратмора время от времени касались беретты, лежавшей у него на коленях. При мысли о том, что Хейл позволил себе прикоснуться к Сьюзан, кровь закипела в его жилах, но он помнил, что должен сохранять ясную голову, Стратмор с горечью признал, что сам отчасти виноват в случившемся: ведь именно он направил Сьюзан в Третий узел. Однако он умел анализировать свои эмоции и не собирался позволить им отразиться на решении проблемы Цифровой крепости. Он заместитель директора Агентства национальной безопасности, а сегодня все, что он делает, важно, как. Его дыхание стало ровным. - Сьюзан.
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Это был агент Колиандер из Севильи. Он перегнулся через плечо Беккера и заговорил в микрофон: - Не знаю, важно ли это, но я не уверен, что мистер Танкадо знал, что он пал жертвой покушения. - Прошу прощения? - проговорил директор. - Халохот был профессионалом высокого уровня, сэр. Мы были свидетелями убийства, поскольку находились всего в пятидесяти метрах от места.
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- Клянусь, я сделаю. Этим я и занимался сегодня весь день - считывал тексты с его терминала, чтобы быть наготове, когда он сделает первый шаг, чтобы вмонтировать этот чертов черный ход. Вот почему я скачал на свой компьютер его электронную почту. Как доказательство, что он отслеживал все связанное с Цифровой крепостью. Я собирался передать всю эту информацию в прессу.
- Он замолчал, не зная, что сказать. Беккер терпеть не мог говорить с автоответчиком: только задумаешься, а тот уже отключился. - Прости, не мог позвонить раньше, - успел сказать .