• 1 - Chromosome Structure
  • 2 - Chromosome Compaction
  • 3 - Chromosome Variation
  • 4 - Gender Identity vs Biological Sex
  • 5 - Nucleic Acid Structure
  • 6 - DNA Replication
  • 7 - Mutations and DNA Repair
  • 8 - Polymerase Chain Reaction (PCR)
  • 9 - Transcription
  • 10 - RNA Modifications
  • 11 - Translation
  • 12 - Gene Cloning
  • 13 - The lac Operon
  • 14 - Gene Regulation in Eukaryotes
  • 15 - Epigenetics
  • 16 - Genome Editing
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  • Translations
  • 13 - The lac Operon

    In Part 13, we will begin learning about the mechanisms that regulate gene expression in bacteria. Gene expression refers to processes that activate structural genes, producing a mRNA molecule by transcription and a functional protein product by translation. Specifically, we will study the expression of the lac operon system in the bacterium E. coli. The lac operon contains the structural genes that produce protein products that function to metabolize lactose for energy production.

    Some bacterial genes are always transcribed. These genes that are always expressed are called constitutive or housekeeping genes. Note that constitutive genes produce constitutive or housekeeping proteins. Housekeeping proteins are required for the normal functioning of the bacterial cell, the so-called housekeeping functions.  The genes that produce the proteins involved in glycolysis are example housekeeping genes. 

    Regulated genes change expression under different environmental conditions. In one environment, the regulated gene is transcribed, while in another environment the regulated gene is silenced.  The mRNAs produced from regulated genes are translated to make inducible proteins. Inducible proteins are tightly controlled so that thousands of copies of the protein may be present in certain environments, while only a few or no copies of the protein are produced in other environments. Regulated genes and their protein products are advantageous because they allow bacteria to adapt to changing environments, competing for available resources, such as carbon or nitrogen. 

    Key Questions

    • What is the difference between a constitutive and a regulated gene?
    • What is one metabolic process that is always occurring in a bacterial cell (i.e., involve housekeeping genes)?

    Inducible Genes

    Gene regulation in bacteria often involves controlling the initiation of transcription. Transcriptional regulation requires the binding of regulatory transcription factor proteins to regulatory DNA sequences near the promoter region of a gene. These regulatory transcription factor proteins function to either enhance or inhibit sigma (σ) factor protein and RNA polymerase core enzyme binding to the promoter.  Regulatory transcription factor proteins include:

    Repressor and activator proteins contain a DNA binding domain and have a binding site for small organic molecules (sugars, amino acids, or nucleotides) called effectors. When an effector molecule binds, the three-dimensional structure of the repressor or activator protein changes.  This change in protein shape influences the ability of the activator protein or repressor protein to bind to the DNA.

    How do bacteria turn a regulated gene from an off state to an on state? For example, how does a bacterium produce the enzymes necessary to metabolize the sugar lactose when lactose becomes available in the environment? An inducer effector molecule causes transcription to increase (figure 13.1). Inducers can function in two different ways:

    Figure 13.1 Inducing a Gene --- Image created by SL

    Key Questions

    • How do repressor and activator proteins affect transcription?
    • What is an effector molecule?
    • Describe two ways that an inducer can activate the transcription of a gene.

    Repressible Genes

    How do bacteria turn a regulated gene from an on state to an off state? For example, how does a bacterium stop producing the enzymes required to make the amino acid tryptophan, when there is plenty of tryptophan in the environment? The presence of an effector molecule inhibits transcription in two ways (figure 13.2):

    Figure 13.2 Repressing a Gene --- Image created by SL

    Key Questions

    • Describe how a corepressor and an inhibitor can turn a transcribed gene to an off state.

    Enzymes Involved in Lactose Metabolism in E. coli

    Now we will turn our attention to a specific example of gene regulation in the bacterium E. coli, involving the regulation of the structural genes involved in lactose metabolism.  Lactose is a sugar that can be used as a carbon and energy source for the bacterium E. coli when the preferred carbon and energy source, glucose, is limited. Lactose breakdown by an E. coli cell involves three enzymes (figure 13.3):

    Figure 13.3 The E. coli enzymes involved in lactose metabolism.  Lactose permease moves lactose across the cytoplasmic membrane into the cytoplasm of the E. coli cell.  β-galactosidase converts lactose into glucose and galactose.  β-galactosidase also forms the inducer allolactose.  The enzyme galactoside transacetylase is not shown in the figure. --- Image created by SL

    Key Questions

    • What are the functions of the three bacterial enzymes involved in lactose breakdown?


    In bacteria, a group of structural genes can be under the control of a single group of regulatory DNA sequences, a single promoter sequence, and a single terminator sequence. This grouping of structural genes is an operon (figure 13.4). The organization of structural genes into operons allows all of proteins involved in a single biochemical pathway (e.g., lactose metabolism) to be regulated in a coordinated way. When an operon is transcribed, a polycistronic mRNA is produced that contains the coding regions for multiple individual proteins.

    Typical operons contain a promoter.  Recall that the promoter serves as the binding site for sigma (σ) factor and contains the transcription start site (+1 site) for the operon. Operons also contain an operator DNA sequence that serves as a repressor protein binding site, an activator binding site where an activator protein binds, structural genes that encode proteins, and a terminator sequence that signals the end of transcription. Recall that transcriptional terminators in bacteria work either using the rho (ρ)-dependent or rho (ρ)-independent mechanism.

    Figure 13.4 Operon Structure --- This image is used from OpenStax (access for free at https://openstax.org/books/biology-2e/pages/1-introduction)

    Key Questions

    • What is an operon?
    • Why is it advantageous to organize structural genes into operons?
    • What is a polycistronic mRNA?
    • What is an operator and an activator binding site?

    The Lactose (lac) Operon

    François Jacob and Jacques Monod first described transcriptional regulation by studying lactose metabolism in E. coli. Jacob and Monod won the Nobel Prize in 1965 for their work.  Lactose metabolism in the bacterium E. coli requires regulating genes within the lactose (lac) operon. The lac operon contains the following DNA sequences and structural genes (figure 13.5):

    Upstream of the lac operon is another structural gene, called lacI, that contains its own promoter and terminator. The lacI gene encodes the lac repressor protein. The lac repressor protein binds to the lacO sequence and turns off the expression of the lac operon (in other words, the lac operon displays negative control via the lac repressor). The lacI gene is a constitutive (housekeeping) gene and is therefore always transcribed.

    Figure 13.5 Lac operon structure --- Image created by Alex Baff. 

    Key Questions

    • What are names of the three structural genes of the lac operon?
    • What are the names and functions of the four regulatory DNA sequences within the lac operon?
    • What is the function of the lacI gene?

    lac Operon Expression

    In the absence of lactose, repression of the lac operon occurs as follows (figure 13.6 and 13.7):

    1. lacI is a constitutive gene, meaning that it is always transcribed. Transcription of the lacI gene produces a lacI mRNA that is then translated to produce the lac repressor protein.
    2. The lac repressor protein binds to the operator (lacO) DNA sequence.
    3. Sigma (σ) factor and the RNA polymerase core enzyme do not bind efficiently to lacP when the lac repressor is bound to lacO. As a result, the three structural genes (lacZ, lacY, and lacA) of the lac operon are transcribed at a low level, producing only a few copies of β-galactosidase, lactose permease, and galactoside transacetylase per cell.

    When lactose becomes available in the environment, the lac operon is induced as follows:

    1. The few copies of lactose permease expressed by E. coli move lactose across the cytoplasmic membrane into the cytoplasm of the cell.
    2. The few copies of β-galactosidase present convert lactose into allolactose.
    3. Allolactose binds to the lac repressor protein. 
    4. The conformation of the lac repressor protein changes.
    5. The lac repressor protein is released from the lacO DNA sequence.
    6. Sigma (σ) factor and the RNA polymerase core enzyme bind to lacP efficiently when the lac repressor protein is released from the DNA.
    7. The structural genes of the lac operon (lacZ, lacY, and lacA) are transcribed to produce polycistronic mRNAs.  The polycistronic mRNAs are then translated to produce thousands of copies each of β-galactosidase, lactose permease, and galactoside transacetylase.
    8. Lactose is metabolized by the E. coli cell.

    Note that when lactose is no longer present in the environment, the lac operon resets.  Allolactose is released from the lac repressor protein, causing the lac repressor to bind once again to lacO.  The β-galactosidase, lactose permease, and galactoside transacetylase proteins in the cytoplasm are eventually degraded.

    Figure 13.6 Lac Operon in the Absence/Presence of Lactose --- Image is borrowed from OpenStax (access for free at https://openstax.org/books/biology-2e/pages/1-introduction) modified by SL
    Figure 13.7 Summary of lac Operon Induction --- Image created by SL

    Key Questions

    • Describe the steps involved in inducing the lac operon.

    The lacI - Strain Displays Constitutive Expression of the lac Operon

    To gain an appreciation of the genetics involved in lac operon regulation, let us turn our attention to the experiment that determined the function of the lacI gene product, which we now know makes the lac repressor protein.  When François Jacob and Jacques Monod first started studying lactose metabolism, they identified a mutant strain of E. coli that they named lacI -. In this lacI - mutant strain, the enzymes involved in lactose metabolism were always produced, even in the absence of lactose. Thus, the lacI - mutation is a constitutive mutation producing constitutive expression of the lac operon. How could this phenotype be explained?

    Jacob and Monod reasoned that the lacI- constitutive phenotype could be explained in two ways:

    Mutant and Merozygote Strains of E. coli

    To distinguish between the two hypotheses indicated above, Jacob and Monod examined two strains of E. coli. Jacob and Monod studied the lacI- strain described above, and they examined an unusual strain of E. coli called a merozygote, or partial diploid.

    Recall that bacteria typically have a single circular chromosome; however, bacteria also contain small circular plasmid DNA molecules in addition to the chromosome. These plasmids are used in gene cloning experiments (see Part 12).  A common type of plasmid is the F plasmid that functions in bacterial fertility (i.e., DNA transfer between bacteria). The merozygote strain that Jacob and Monod examined in their experiments contained a modified F plasmid (F’ plasmid), which contained a lacI gene and the lac operon. Thus, E. coli cells that contain an F’ plasmid are merozygotes (partial diploids), containing a copy of lacI and the lac operon genes on both the chromosome and on the F’ plasmid.

    The merozygote strain used by Jacob and Monod contained lacI- on the chromosome and a wild-type copy of the gene (lacI +) on the F’ plasmid; this E. coli strain was in essence a lacI +/lacI - heterozygote. The other DNA sequences within the lac operon (lacP, lacO, lacZ, lacY, and lacA) were wild-type and were found on both the chromosome and the F’ plasmid.  Thus, the E. coli merozygote strain was homozygous for lacP, lacO, lacZ, lacY, and lacA.

    Key Questions

    • What is a merozygote?
    • Why might it be advantageous for an E. coli cell to have two copies of each lac operon gene?

    The Jacob and Monod Experiment

    The Jacob and Monod experiment compared a lacI - strain (lac operon is always expressed) to the lacI +/lacI - merozygote strain. The experiment was done as follows:

          1. The mutant (lacI -) and the merozygote (lacI +/lacI -) strains were grown in separate flasks. 

          2. Each bacterial culture was then split into two smaller flasks, a control flask and an experimental flask.  For example:

         3.  Lactose was added to the experimental reactions (flasks 2 and 4).

         4. The bacterial cultures were incubated to allow transcription of the lac operon and translation of the β- galactosidase, lactose permease, and galactoside transacetylase proteins.

         5. The bacterial cells in each flask were then lysed to release the β- galactosidase, lactose permease, and galactoside transacetylase proteins.

         6. The β-galactosidase levels in each of the four lysates were measured. β-galactosidase can convert the chemical β-O-nitrophenylgalactoside (β-ONPG), which is colorless, into galactose and O-nitrophenol, which is yellow. Note that if a yellow product is formed, β-galactosidase is present (i.e. the lac operon was transcribed).

         7. The O-nitrophenol (yellow product) levels in each lysate were measured using a spectrophotometer.

    Key Questions

    • Explain the purpose of flasks 1–4 in the Jacob and Monod experiment.
    • Explain what is occurring when a reaction turns yellow.

    lacI + Produces a Diffusible Repressor Protein

    Yellow color was observed in flasks 1 and 2 for the mutant E. coli strain (lacI -). Thus, in the lacI - strain, β-galactosidase is produced in the absence and in the presence of lactose (i.e., expressed constitutively).

    In the merozygote strain, no yellow color was produced in the absence of lactose (flask 3); however, two times the yellow product was produced in the presence of lactose (flask 4). This means that β-galactosidase is not produced when lactose is absent because the lacI + gene on the F’ plasmid produces a protein (i.e., the lac repressor protein) that binds to both copies of lacO in the cell, and thus inhibits expression of both the chromosomal and F’ plasmid genes that make β-galactosidase. Remember that bacteria do not have a nuclear membrane, so both the host chromosome and the F' plasmid are found in the cytoplasm. This lac repressor protein diffuses throughout the cytoplasm of the cell and can bind to any lacO sequence. Because the lac repressor can bind to any operator in the cell, the lac repressor is said to be an example of a trans-acting factor.

    When lactose is present, lactose is converted to allolactose, and allolactose releases the lac repressor proteins from both copies of lacO. The lac operons on both the chromosome and on the F’ plasmid are now expressed (flask 4). The expression of two copies of the lacZ gene produces two times as much β-galactosidase protein, leading to the production of two times the yellow product in flask 4.

    This experimental result provided supporting evidence for the defective repressor hypothesis. It is worth noting that in the case of the lacI - constitutive activator hypothesis, the lac operon would have been expressed by the merozygote strain in both the absence (flask 3) and in the presence of lactose (flask 4).  Thus, both flasks 3 and 4 should have produced two times the yellow color at the conclusion of the experiment.

    Key Questions

    • Explain how the flask 3 result showed that the lac repressor protein is an example of a trans-acting factor.

    Glucose and the lac Operon

    The lac operon can also be regulated by glucose. Glucose is the preferred carbon and energy source used by E. coli, so the genes involved in glucose breakdown (catabolism) are expressed constitutively (always transcribed). In the presence of glucose, the lac operon is not needed, so transcription of the lac operon is turned off (so-called catabolite repression). When glucose levels decrease and lactose is present, this catabolite repression is alleviated, and the lac operon is transcribed. Lactose is then used by the E. coli cell as the carbon and energy source.  The sequential use of sugars—first glucose, followed by lactose—is called diauxic growth (figure 13.8).

    Figure 13.8 Diauxic growth of E. coli.  E. coli uses glucose first, then switches to using lactose. --- Image created by SL

    How is the lac operon repressed (turned off) by glucose? Glucose repression of the lac operon involves a(n):

    cAMP is produced from ATP by the enzyme adenylyl cyclase. When glucose is present in the environment, adenylyl cyclase activity is inhibited, and cellular cAMP levels are low. When glucose levels in the environment are low, adenylyl cyclase activity increases, resulting in higher levels of cAMP in the cell.

    When the CAP protein binds to cAMP, the CAP protein changes conformation (shape) and can then bind to the CAP site in the DNA.  Lac operon transcription is activated. In fact, for sigma (σ) factor and the RNA polymerase core enzyme to bind efficiently to the lac promoter and transcribe the lac operon, CAP must be bound to the CAP site.

    Figure 13.9 Cyclic AMP (cAMP) structure

    Key Questions

    • Describe how the lac operon responds when glucose levels are high or low (assuming that lactose is present).
    • What is the function of adenylyl cyclase?
    • Explain the relationship between cAMP levels and CAP function.
    • Describe the relationship between CAP binding to the CAP site and the transcription of the lac operon.

    Regulation of the lac Operon by the lac Repressor and CAP

    The interaction between a positive regulatory signal (CAP) and a negative regulatory signal (the lac repressor protein) makes transcriptional regulation of the lac operon more complicated. What happens to lac operon expression when an E. coli cell encounters the following environmental conditions (figure 13.10)?

    In summary, there is only one way the lac operon is transcribed efficiently: glucose must be absent from the environment, and lactose must be present.

    Figure 13.10 Lac Operon Expression Under All Cellular Conditions --- Image created by SL

    Key Questions

    • Why is the lac operon off if neither glucose nor lactose are present in the cell?
    • Why is the lac operon off if glucose is present and lactose is absent in the cell?
    • Why is the lac operon off if both glucose and lactose are present in the cell?
    • Why is the lac operon on if glucose is absent and lactose is present in the cell?

    Other Gene Regulation Mechanisms in Bacteria

    The lac operon is one example of how bacteria can control gene expression. There are many ways to control the expression of a gene in bacteria, including regulating transcription, translation, and the protein product produced following translation.

          Key Questions

          • What is an advantage of transcriptional regulation?
          • What is an advantage of posttranslational regulation?

          Review Questions

          Fill in the blank:

          1. An _________________ protein is a type of regulatory transcription factor that increases transcription.
          2. _________________ molecules are organic compounds that regulate the functions of repressor and activator proteins.
          3. ___________________________ is the enzyme that catalyzes the cleavage of the disaccharide lactose into two simple sugars.
          4. Identify each of the following genes of the lac operon:
          1. The DNA binding site for the lac repressor protein is called _______________________.
          2. The two inducers of the lac operon are ________________ and ________________.
          3. A merozygote contains two copies of a gene; one gene is located on the ______________ while the other gene is located on the ________________.
          4. _____________________ is an enzyme that is inhibited by glucose.
          5. Lactose and glucose regulate expression of the lactose operon. The highest expression of the lac operon occurs when ________________ is absent and __________________ is present.

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