lac Operon

10 Key excerpts on "lac Operon"

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  Current Topics in Cellular Regulation

  • Bernard L. Horecker, Earl R. Stadtman(Authors)
  • 2014(Publication Date)
  • Academic Press

    (Publisher)

  One is transcriptional control which involves regulation of the transcription of DNA to yield mRNA. The other is translational control which in-volves synthesis of protein using the message coded in mRNA. In this article, we focus our discussion on the first type of control—the regula-tion of gene expression by transcriptional control. The lactose (lac) operon of Escherichia coli has long been an impor-tant model for developing an understanding of the control of gene ex-pression. In fact, Jacob and Monod's (39) formulation of their well-known hypothesis of operon structure and its role in the regulation of protein synthesis was mainly based on information from the lac ope-ron. In their hypothesis, an operon is defined as a group of functionally related structural genes which can be turned on or off coordinately by the same regulatory switch. The operator locus is visualized as the switch which controls the transcription of the genes in the operon. The transcript (mRNA) is then translated to yield a specific protein se-quence. Over the years, both important principles and specific details of cellular regulatory mechanisms have been established by analyzing the lac Operon. B. The lac Operon The main features of the lac Operon are shown in Fig. 1. In this figure, z, y, and a are the structural genes which code for /3-galactosidase, permease, and transacetylase, respectively. The i gene is a regulatory gene which codes for the synthesis of the lac repressor, p is the promoter site recognized by the DNA-directed RNA polymerase to initiate mRNA synthesis, o is the operator site, a region on the DNA molecule which is specifically recognized by the lac repressor. In the absence of an inducer, the repressor protein binds to the operator and prevents transcription of the structural genes z, y, and a.

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  Microbe

  • Michele S. Swanson, Elizabeth A. Joyce, Rachel E. A. Horak(Authors)
  • 2022(Publication Date)
  • ASM Press

    (Publisher)

  Why does the cell maintain a small amount of the Lac proteins? Once lac- tose becomes available, these basal protein levels equip the cell to take up and metabolize some of this sugar, an event that activates the lac Operon (Fig. 11.6B). Lactose is brought into the cells by LacY permease and is con- verted into a metabolite called allolactose by the β-galactosidase LacZ. This derivative of lactose is the actual inducer of the lac Operon: it binds the LacI repressor, triggering a conformational change that releases this allo- steric regulatory protein from its promoter. RNAP is now free to make lacZYA transcripts. More of the operon products are made, and the cells bring more lactose in. If lactose is all the cells need to make the inducer that removes the repressor of the lac Operon, why in the case study did E. coli grow with lactose only after glucose was consumed? Later in the chapter, we will learn the answer: it is due to an energy-conserving process called catabolite repression. Research on the lac Operon of enteric bacteria generated a number of fundamental concepts about operons, including the following. 1. Initiation of transcription from the promoter of an operon can be a site of regulation. 2. Initiation of transcription can be controlled by allosteric proteins (i.e., the protein’s activity is governed by the binding of specific ligands). 3. Increase in the expression of an operon can be triggered by relief of a nega- tive control. The lac Operon is an elegant example of how microbes regulate a metabolic adaptation, but it is not the only mechanism. Microbes exhibit a dazzling diver- sity of solutions to metabolic and physiologic challenges. In general, operons differ as to (i) the site at which control is exerted, (ii) the mode of control (positive or negative), and (iii) the molecular device used to bring about the regulation.

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  Fundamental Molecular Biology

  • Lizabeth A. Allison(Author)
  • 2021(Publication Date)
  • Wiley-Blackwell

    (Publisher)

  Why are genes organized into operons? Bacteria need to respond swiftly to changes in their environment, to switch from metabolizing one substrate to another quickly and energetically efficiently. When glucose is abundant, bacteria use it exclusively as their food source, even when other sugars are present. However, when glucose supplies are depleted, bacteria have the ability to rapidly take up and metabolize alternative sugars, such as lactose. The synthesis of enzymes in response to the appearance of a specific substrate – a pro-cess called induction – is a widespread mechanism in bacteria and single-celled eukaryotes such as yeast. Characterization of the lac repressor In addition to requiring an RNA intermediate, the Jacob–Monod model for gene regulation also proposed the existence of a repressor protein. Between 1966 and 1972, it was shown that both Lac and λ repressors are indeed proteins. They bind to operator DNA adjacent to the promoter and inhibit the capacity of RNA polymerase to transcribe. Since a bacterial cell contains only 10–20 copies of the lac Operon repressor, its detection and isolation in 1966 by Walter Gilbert and Benno Müller-Hill was a remarkable accomplishment, when many sensitive technologies in use today were not available. To determine whether Lac repressor bound to operator DNA , Gilbert and Müller-Hill carried out an in vitro –binding assay. Before the advent of gene cloning techniques, stud-ies of bacterial genes had to rely on bacteriophage variants that had incorporated pieces of bacterial DNA (see Figure 13.1). Conveniently, a phage strain was available that included lac Operon DNA. Gilbert and Müller-Hill mixed radioactively labeled purified Lac repres-sor with phage phi ( ϕ ) 80 DNA that had incorporated the lac operator or with phage ϕ 80 lacking the lac operator (Figure 8.13).

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  Biophysics for Beginners

  A Journey through the Cell Nucleus

  • Helmut Schiessel(Author)
  • 2013(Publication Date)
  • Jenny Stanford Publishing

    (Publisher)

  The lac repressor considered here controls the transcription of three genes called lacZ , lacY and lacA . These genes encode for proteins that are involved in the metabolism of milk sugar, the so-called lactose. How do E. coli bacteria get into contact with lactose? Since E. coli live in our intestines, they will find themselves surrounded by lactose whenever we drink a glass of milk. Lactose is made from two covalently bound sugars, galactose and glucose. As a first step in the metabolism, the lactose needs to be broken into these two components. This is done by the enzyme β -galactosidase. This protein is encoded in the gene lacZ . lacY encodes for β -galactoside permease, a membrane protein that pumps lactose into the cell, and lacA encodes for β -galactoside transacetylase that is required for the chemical modification of the sugar molecules. As shown in Fig. 8.1 to the left of the lac Operon, in the upstream direction, there is an operator site which is the binding site for the lac repressor. Adjacent to that site is the promoter, the site where the RNA polymerase has to bind first before it starts to transcribe the genes. If the operator site is unoccupied, the polymerase can bind and transcribe the three genes downstream, see Fig. 8.1(a). If the lac repressor is bound to the operator, RNA polymerase is blocked from binding to the promoter and the genes cannot be transcribed, see Fig. 8.1(b). The gene of the lac repressor itself, lacI , lies nearby the lac Operon and is always expressed at a moderate level. This ensures that there are always lac repressors present in the cell even though proteins are broken down after a certain period of time. The task of the lac repressor is to keep the lac Operon inaccessible as long as there is no milk sugar around and to allow transcription if it is present. That way the lac repressor ensures that the cell does not waste energy in producing the proteins for the lactose metabolism in the absence of lactose.

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  Microbiology

  • Dave Wessner, Christine Dupont, Trevor Charles, Josh Neufeld(Authors)
  • 2016(Publication Date)
  • Wiley

    (Publisher)

  lacI, is located upstream of the operon and encodes the LacI repressor protein, which controls the operator.

  Figure 11.4.

  Action of β-galactosidase

  Lactose can be cleaved into glucose and galactose that can be further metabolized by the cell. Lactose can also be converted to allolactose, which can turn on the lac operon.

  How can the cell regulate the expression of these structural genes? A single promoter controls the initiation of transcription in an operon. Regulatory proteins that bind the operator, along with associated effector molecules, modulate the ability of RNA polymerase to bind to the promoter and initiate transcription ( Table 11.2 ) . The genes encoding these regulatory proteins may be located adjacent to the operon on the chromosomal DNA, or they can be located elsewhere on the chromosome. As we will see in this section, these regulatory proteins can inhibit operon transcription, a process referred to as negative control, or facilitate operon transcription, a process referred to as positive control. Additionally, as we will see in Section 11.3

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  Transcription

  • William M. Brown, Philip M. Brown(Authors)
  • 2001(Publication Date)
  • CRC Press

    (Publisher)

  I gene acted as a represser controlling the production of the three proteins. The experiments conducted used cells containing two copies of each gene, with at least one being active, and relied not on molecular biology techniques, but on bacterial physiology to accomplish this. The first copy in each case was located on the bacterial chromosome; the second copy was introduced into the bacterium on a plasmid (an extrachromosomal piece of DNA) by a process known as F-duction.

  Once the plasmid is introduced into the cell, the cell is effectively diploid for the genes in question. By combining different mutant strains and different F’plasmids, Jacob and Monod were able to form different combinations of native and mutated genes and to deduce how the system operated.

  In the rest of this chapter we will first describe how the lactose operon is now known to be oriented and controlled and then explain the experimental design that led to the elucidation of this mechanism.

  4.2The structure and mechanism of the lactose operon

  An “operon” is a set of functionally related bacterial genes under common control. The lactose operon of E. coli encodes proteins essential to the bacterium in metabolising lactose.

  In bacteria, genes encoding proteins involved in the same process are often found immediately adjacent to each other; in addition to being physically close or adjacent, regulation of expression of these genes is such that they are all turned on or off together, that is, they are regulated in a co-ordinated manner. Such a group of co-ordinately regulated genes, together with the control elements, is referred to as an operon. Such a grouping of related genes under a common control mechanism permits the bacteria to adapt rapidly to changes in the environment, including the availability of a carbon source or the absence of a necessary ammo acid.

  In contrast to the control mechanisms seen in eukaryotes, negative control of expression of bacterial operons is the norm.

  The lactose operon of E. coli encodes proteins involved in the metabolism of lactose. Under normal conditions the operon is only induced when lactose is available. The operon itself is known to contain three active genes denoted by the letters Z, Y and A. The Z gene encodes -galactosidase, the gene encodes a permease, and the A gene encodes a transacetylase enzyme. These genes lie adjacent to each other in the E. coli genome in the order 5’-Z- Y-A. Preceding these genes there are two other important regions, the operator (O) and the promoter (P) (indicated in Figure 9.2 ). The operator acts as a switch for the activation of the operon and the promoter is the site from which gene transcription starts. In contrast to the control mechanisms seen in eukaryotes (discussed in chapter 4 ), negative

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  Microbiology

  • Dave Wessner, Christine Dupont, Trevor Charles, Josh Neufeld(Authors)
  • 2020(Publication Date)
  • Wiley

    (Publisher)

  Reproduced with permission of John Wiley & Sons. 408 CHAPTER 12 Regulation of Gene Expression in turn, cannot bind to the lac Operon promoter, and transcrip- tion of the lac Operon genes does not occur (Figure 12.7). This phenomenon, called catabolite repression, ensures that E. coli preferentially utilizes glucose. Conversely, when glucose levels are low, cAMP levels increase, CRP facilitates binding of RNA polymerase to the promoter, and the cell can make the enzymes needed to use lactose (see Figure 12.7b). Of course, in the absence of lactose, allolactose would not be present to remove the repressor from the operator site and the enzymes would be produced only in very small amounts (Figure 12.8). Investigating the lac Operon In the preceding two sections, we gained a better understand- ing of how the lac Operon is regulated. In this section, we will address a different question about this operon. How do we know that the operon is regulated in these ways? The nature To better understand how positive control works, let’s revisit the lac Operon. This operon exhibits both negative and positive transcriptional control. E. coli cells grow better using glucose than using lactose. Quite simply, cells expend less energy during glucose metabolism as it enters glycolysis directly. Conversely, lactose must first be converted to galac- tose and glucose. As a consequence, the presence of glucose overrides the ability of lactose to activate the lac Operon. As we noted earlier, the presence of lactose in an E. coli cell leads to the removal of a repressor from the operator, allowing RNA polymerase to bind. For transcription of the lac Operon to occur, however, an activator protein (cAMP receptor protein, or CRP) must bind to the activator binding site to enhance binding of RNA polymerase to the promoter. Further- more, CRP requires the coactivator cyclic AMP, or cAMP, before it can bind to the activator binding site. In the presence of glu- cose, levels of cAMP are low.

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  Snyder and Champness Molecular Genetics of Bacteria

  • Tina M. Henkin, Joseph E. Peters(Authors)
  • 2020(Publication Date)
  • ASM Press

    (Publisher)

  Cellular and Molecular Biology, 2nd ed. (ASM Press, Washington, DC, 1996).

  Catabolite Regulation of the lac Operon

  In addition to being under the control of its own specific repressor, the lac operon is regulated in response to the availability of other carbon sources through catabolite repression . Catabolic pathways are used to break down substrates to yield carbon or energy. The catabolite repression system ensures that the genes for lactose utilization are not expressed if a better carbon and energy source, such as glucose, is available. The name “catabolite repression” is a misnomer, at least in E. coli, since the expression of operons under catabolite control in E. coli requires a transcriptional activator, the catabolite activator protein (CAP, historically called Crp, for catabolite repression protein), and the small-molecule effector cyclic AMP (cAMP). Many operons are under the control of CAP-cAMP, and we defer a detailed discussion of the mechanism of catabolite regulation until chapter 12 , since it is a type of global regulation.

  Structure of the lac Control Region

  Figure 11.6 illustrates the structure of the lac control region in detail, showing the nucleotide sequences of the lac promoter and two of the operators (o1 and o3 ), as well as the region to which CAP binds. The lac promoter is a typical σ70 bacterial promoter with characteristic −10 and −35 regions (see chapter 2 ). One of the operators to which the LacI repressor binds (o1 ) overlaps the initiation site (+1 in Figure 11.6A ) for transcription of lacZ, lacY, and lacA. The other lac operator sequences are positioned upstream and downstream of the promoter, and the sequences of all three operator sites are shown in Figure 11.6B . Each symmetrical half of an operator binds a LacI monomer. Simultaneous binding of the LacI tetramer to two operator sites (e.g., o1 and o3 , as shown in Figure 11.5B

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  Further Milestones in Biochemistry

  • M.G. Ord, L.A. Stocken(Authors)
  • 1997(Publication Date)
  • Elsevier Science

    (Publisher)

  An experiment in which part of the lactose operon was fused by deletion of intervening sequences illustrated the specificity of the DNA binding site for its regulator protein (Jacob et al., 1965). On one side the deletions extended into the z gene and on the other side into part of the operon for the biosynthesis of purines. These deletions resulted in new hybrid operons and with several of these the j8-galactoside permease and the transacetylase were no longer induced by lactose but were repressed by purines. Gene fusions in vitro were to provide a valuable tool for testing gene expression. Figure 7 is a slightly modified version of the original Model 1 and it can be seen that while the operator remains the site of repressor binding the the site at which transcription is initiated is identified as the promoter. The picture was rounded off by the isolation of purified lac Operon DNA. (Shapiro et al., 1969). The lac genes were fused with bacteriophages k and <^80 to increase the amount of DNA produced. The DNA of the two phages was separated into single strands, hybridized, and the nonhomologous single-stranded ends removed. The resulting double-stranded DNA was the purified lac Operon that could be seen under the electron microscope (Figure 8). OTHER OPERONS The lac Operon model was so satisfying and elegant that it dominated thinking about the regulation of gene expression for a long time. Almost everyone interested in bacterial enzymes had accepted that negative control by repressors of structural genes could be a universal method for regulation of protein synthesis. It was tacitly assumed that most, if not all, inducible and repressible enzyme systems were similar to that for /3-galactosidase. However, it is interesting to reflect that Monod had originally seen the inducer playing a positive role in enzyme induction. Before becoming committed to j8-galactosidase he had been interested in amylomaltase (Monod and Torriani, 1950).

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  Mathematical Concepts and Methods in Modern Biology

  Using Modern Discrete Models

  • Raina Robeva, Terrell Hodge(Authors)
  • 2013(Publication Date)
  • Academic Press

    (Publisher)

  lac operon. Thus, at a minimum, our model should show that the operon has two steady states, On and Off. When extracellular glucose is available, the operon should be Off. When extracellular glucose is not present and extracellular lactose is, the operon must be On. We next demonstrate that our model satisfies these conditions.

  Recall that the operon is On when mRNA is being produced . When mRNA is present, the production of lac permease, and -galactosidase is also turned on. This corresponds to the fixed-point state . On the other hand, when mRNA is not made, the operon is Off. This also means no production of lac permease, and -galactosidase. This corresponds to the fixed-point state .

  For the Boolean model of the lac operon from Eqs. (1.4) , there are four possible combinations for the values and of the model parameters: ; and . For each one of these pairs of values we can determine the state space transition diagram of the model from the update functions in Eqs. (1.4) . The results are shown in Figure 1.7 . Notice that according to the model, the operon is On only when external glucose is unavailable and external lactose is present. In all other cases, the operon is Off. These model predictions reflect exactly the expected behavior of the lac operon based on the underlying regulatory mechanisms described earlier. This means that our initial model is capable of describing the most fundamental behavior of the lac operon system and captures the main qualitative properties of lac operon regulation.

  Exercise 1.8

  Verify that the space state diagram for the Boolean model described by Eqs. (1.4) is as presented in Figure 1.7b . Notice that for some values of the parameters, the transition functions simplify significantly when we apply short-circuit evaluation for the appropriate Boolean expressions. For instance, when , the equations for the transition functions will be: , regardless of the values of L and and , regardless of the values of L , E , and . 

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