Jacob and Monod worked out the structure and function of the lac operon by analyzing mutations that affected lactose metabolism. To help define the roles of the different components of the operon, they used partial diploid strains of E. coli. The cells of these strains possessed two different DNA molecules: the full bacterial chromosome and an extra piece of DNA. Jacob and Monod created these strains by allowing conjugation to take place between two bacteria (see Chapter 7). In conjugation, a small circular piece of DNA (the F plasmid, see Chapter 7) is transferred from one bacterium to another. The F plasmid used by Jacob and Monod contained the lac operon, so the recipient bacterium became partly diploid, possessing two copies of the lac operon. By using different combinations of mutations on the bacterial and plasmid DNA, Jacob and Monod determined that some parts of the lac operon are cis acting (able to control the expression of genes only when on the same piece of DNA), whereas other parts are trans acting (able to control the expression of genes on other DNA molecules).

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STRUCTURAL-GENE MUTATIONS Jacob and Monod first discovered some mutant strains that had lost the ability to synthesize either β-galactosidase or permease. (They did not study the effects of mutations on the transacetylase enzyme in detail, so transacetylase will not be considered here.) The mutations in those mutant strains mapped to the lacZ or lacY structural genes and altered the amino acid sequences of the proteins encoded by these genes. These mutations clearly affected the structure of the proteins, but not the regulation of their synthesis.

Through the use of partial diploids, Jacob and Monod were able to establish that mutations at the lacZ and lacY genes were independent and usually affected only the product of the gene in which the mutation occurred. Partial diploids with lacZ⁺ lacY⁻ on the bacterial chromosome and lacZ⁻ lacY⁺ on the plasmid functioned normally, producing β-galactosidase and permease in the presence of lactose. (The genotype of a partial diploid is written by separating the genes on each DNA molecule with a slash: lacZ⁺ lacY⁻/lacZ⁻ lacY⁺.) In this partial diploid, a single functional β-galactosidase gene (lacZ⁺) is sufficient to produce β-galactosidase; whether the functional β-galactosidase gene is coupled to a functional (lacY⁺) or a defective (lacY⁻) permease gene makes no difference. The same is true of the lacY⁺ gene.

REGULATOR-GENE MUTATIONS Jacob and Monod also isolated mutations that affected the regulation of protein production. Mutations in the lacI gene affected the production of both β-galactosidase and permease because the genes for both proteins are in the same operon and are regulated coordinately by the lac repressor protein.

Some of these mutations were constitutive mutations, causing the lac proteins to be produced all the time, whether lactose was present or not. Such mutations in the regulator gene were designated lacI⁻. The construction of partial diploids demonstrated that a lacI⁺ gene is dominant over a lacI⁻ gene; a single copy of lacI⁺ (genotype lacI⁺/lacI⁻) was sufficient to bring about normal regulation of protein production. Furthermore, lacI⁺ restored normal control to an operon even if the operon was located on a different DNA molecule, showing that lacI⁺ can be trans acting. A partial diploid with genotype lacI⁺ lacZ⁻/lacI⁻ lacZ⁺ functioned normally, synthesizing β-galactosidase only when lactose was present (Figure 12.9). In this strain, the lacI⁺ gene on the bacterial chromosome was functional, but the lacZ⁻ gene was defective; on the plasmid, the lacI⁻ gene was defective, but the lacZ⁺ gene was functional. The fact that a lacI⁺ gene could regulate a lacZ⁺ gene located on a different DNA molecule indicated to Jacob and Monod that the lacI⁺ gene product was able to operate on either the plasmid or the chromosome.

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Some lacI mutations isolated by Jacob and Monod prevented transcription from taking place even in the presence of lactose. These mutations were referred to as superrepressors (lacI^s), because they produced defective repressors that could not be inactivated by an inducer. The lacI^s mutations produced a repressor with an altered inducer-binding site, which made the inducer unable to bind to the repressor; consequently, the repressor was always able to attach to the operator and prevent transcription of the lac genes. Superrepressor mutations were dominant over lacI⁺: partial diploids with genotype lacI^s lacZ⁺/lacI⁺ lacZ⁺ were unable to synthesize either β-galactosidase or permease, whether or not lactose was present (Figure 12.10).

OPERATOR MUTATIONS Jacob and Monod mapped a second set of constitutive mutations to a site adjacent to lacZ. These mutations occurred at the operator and were referred to as lacO^c (“O” stands for operator and “c” for constitutive). The lacO^c mutations altered the sequence of DNA at the operator so that the repressor protein was no longer able to bind to it. A partial diploid with genotype lacI⁺ lacO^c lacZ⁺/lacI⁺ lacO⁺ lacZ⁺ exhibited constitutive synthesis of β-galactosidase, indicating that lacO^c is dominant over lacO⁺.

Analyses of other partial diploids showed that the lacO gene is cis acting, affecting only genes on the same DNA molecule. For example, a partial diploid with genotype lacI⁺ lacO⁺ lacZ⁻/lacI⁺ lacO^c lacZ⁺ was constitutive, producing β-galactosidase in the presence or absence of lactose (Figure 12.11a), but a partial diploid with lacI⁺ lacO⁺ lacZ⁺/lacI⁺ lacO^c lacZ⁻ produced β-galactosidase only in the presence of lactose (Figure 12.11b). In the constitutive partial diploid (lacI⁺ lacO⁺ lacZ⁻/lacI⁺ lacO^c lacZ⁺; see Figure 12.11a), the lacO^c mutation and the functional lacZ⁺ gene were present on the same DNA molecule, but in lacI⁺ lacO⁺ lacZ⁺/lacI⁺ lacO^c lacZ⁻ (see Figure 12.11b), the lacO^c mutation and the functional lacZ⁺ gene were on different molecules. Thus, the lacO^c mutation affects only genes to which it is physically connected, as is true of all operator mutations. Such mutations prevent the binding of a repressor protein to the operator and thereby allow RNA polymerase to transcribe genes on the same DNA molecule. However, they cannot prevent a repressor from binding to normal operators on other DNA molecules. Watch Animation 12.1 to observe the effects of different combinations of lacI and lacO mutations on the expression of the lac operon. TRY PROBLEM 26

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PROMOTER MUTATIONS Mutations affecting lactose metabolism have also been isolated at the promoter; these mutations, designated lacP⁻, interfere with the binding of RNA polymerase to the promoter. Because this binding is essential for the transcription of the structural genes, E. coli strains with lacP⁻ mutations don’t produce lac proteins either in the presence or in the absence of lactose. Like operator mutations, lacP⁻ mutations are cis acting and thus affect only genes on the same DNA molecule. The partial diploid lacI⁺ lacP⁺ lacZ⁺/lacI⁺ lacP⁻ lacZ⁺ exhibits normal synthesis of β-galactosidase, whereas lacI⁺ lacP⁻ lacZ⁺/lacI⁺ lacP⁺ lacZ⁻ fails to produce β-galactosidase whether or not lactose is present.