Lac operon

The bacterial cells such as E. coli show specific mechanisms for regulating the expression of the genes. They involve a group or cluster genes (operons) exclusively working on the process of regulation. A regulatory control helps the organism to survive and adapt to the environmental changes. The genes present in the bacterial cells mainly get involved in controlling the growth, reproduction, and development. Hence, each of them requires regulatory mechanisms. The regulated genes help in responding to the needs of the cells. These gene products essentially play a role in normal functioning. Always active genes or the housekeeping genes ensure proper synthesis of the products such as enzymes required for protein synthesis. The cells respond to the changes and switch certain genes on or off. An inducible gene consists of a promoter sequence, a controlling site, a coding sequence, and a terminator sequence. Induction of the coding region leads to the transcription and translation. Two key molecules include an inducer and a repressor. The former one helps in the expression of the inducible genes. The latter one represses its expression.

Mostly, the E coli cells utilize glucose as the source of energy. Certain genes (constitutive genes) synthesize the enzymes that help in utilizing this carbon source. However, instead of glucose, if lactose becomes the carbon source, a different set of genes synthesizing lactose metabolism enzymes get involved. The key event for the activation of such genes requires the presence of lactose. This sugar is a disaccharide consisting of two key sugars such as glucose and galactose. The key enzymes involved in the lactose metabolism include beta-galactosidase, lactose permease, and transacetylase. The breakdown of lactose to glucose and galactose occurs due to the activity of the beta-galactosidase enzyme. It also catalyzes the isomerization of lactose to allolactose. The lactose gets transported in the presence of a specific enzyme or a protein. It is known as M protein or lactose permease. Although the function of the transacetylase is not well understood, it gets involved in the lactose metabolism. Coordinate induction of the three genes occurs when the lactose acts as a carbon source. Otherwise, these genes remain inactive. The genes got their names as LacZ, lacy, and LacA for the enzymes beta-galactosidase, lactose permease, and transacetylase respectively.

Experiments:                                                            

·       Mutations in the protein-coding genes:

The wild-type E coli strains produce all three enzymes such as beta-galactose, permease, and transacetylase. However, the nonsense mutants do not produce permease and transacetylase. Also, the beta-galactosidase protein gets incompletely synthesized. Such types of mutations exhibit the polar effect. A polycistronic mRNA consists of all the three enzyme-coding genes. Due to the nonsense mutations in the LacZ gene, the ribosomes no longer slide towards the LacY or LacA genes.

·        Mutations affecting the regulation of gene expression:

Jacob and Monod identified the organization of lac genes in E coli. They identified two main types of genes other than the lac structural genes. These genes included the operator and the repressor genes named as LacO and LacI respectively. Mutations in the operator (Lac O^c) helped the experimenters in studying the regulation of gene expression. They used a partial diploid strain with F’ lacO⁺ lacZ^- lacY⁺ / lac O^c lacZ⁺ lacY^- gene arrangement. One of them has a normal operator, a mutated lac Z gene, and a normal lacY gene. The other region has a constitutive operator, a normal lac Z gene, and a mutated lacY gene. In the absence of the inducer, the beta-galactosidase got synthesized actively. Synthesis of permease was also possible. However, due to mutations in the gene, it got inactively synthesized. The active permease gets synthesized in the presence of lactose and allolactose. Hence, the lac O^c mutations affect the genes downstream.

The LacI mutation studies also utilized the partial diploids with two gene sets. One of them had lacI⁺ Lac O⁺ LacZ^- LacY⁺ indicating the presence of a normal lac I repressor gene, a normal operator gene, a mutated beta-galactosidase synthesizing gene, and a normal permease synthesizing gene respectively. The lacI⁺ is a repressor gene and thus produces a repressor molecule. Both permease and beta-galactosidase got synthesized only in the presence of an inducer. The lacI⁺ gene overcomes the defective gene mutation. Promoter genes are present at the end of the lacZ gene. Mutations in these genes affect all the three structural genes.

Operon model for the Lac genes:

Operator-repressor interactions regulate the cluster of genes known as the operons. The constitutive expression of the repressor gene leads to the transcription of the mRNA. It gives rise to repressor proteins capable of binding to the operator gene. Once bound to an operator, a repressor blocks the transcription of the lac structural genes. The repressor-operator interactions do not allow the RNA polymerase to bind and transcribe the genes. It is an example of the negative control of lac operon. The regulation of lac operon occurs differently in the presence of the lactose. Some of the lactose molecules get converted into allolactose molecules. The allolactose is capable of binding to the repressor protein. Once bound to the repressor, the allolactose changes the conformation of the repressor protein. Hence, the repressor cannot bind to the operator. In such a case, the RNA polymerase binds and transcribes the lac structural genes. Hence, the enzymes beta-galactosidase, permease, and transacetylase get synthesized.

·        Lac O^c mutations: Consider a partial diploid in the absence of the inducer allolactose. The partial diploid consists of lac O⁺ and Lac O^c genes respectively. The transcription and translation of lacI⁺ lead to the formation of repressor proteins. In the case of the lac O⁺ gene, the repressor binds effectively. Hence, no transcription of the lac structural genes occurs. The lac O^c mutations do not allow the binding of the repressor proteins. The polycistronic mRNA having lac O^c gene shows three main structural genes such as normal lac Z gene (lac Z+), a defective lacY gene (lacY-), and a normal lac A gene (LacA+). The repressor proteins do not bind to the operator. Hence, the expression of the lac structural genes occurs. Consider a partial diploid when the inducer allolactose binds to the repressor. The partial diploid consists of two sets of genes. One of them shows the presence of Lac O⁺ Lac Z^- Lac Y^+, and LacA⁺ genes. The other set of genes includes Lac O^c Lac Z⁺ LacY^- and Lac A⁺. The LacO+ interacts with the repressor and leads to the expression of lac structural genes. The LacO^c cannot interact with the repressor. Hence, the RNA polymerase transcribes the lac structural genes.

      

              Image 2: Lac O mutations

·    Lac I^- mutations: Consider a haploid strain. It has normal lac structural genes, a normal operator gene, and a mutated repressor gene. Due to LacI^- mutations, the mutant repressor protein cannot bind to the operator. Hence, the lac structural genes get expressed. Consider a partial diploid with two sets of genes. One of them has a LacI⁺ LacO⁺ lacZ^- LacY⁺ LacA⁺ genes respectively. The other set consists of LacI^- LacO⁺ Lac Z⁺ LacY^- LacA⁺. In either of the cases, the wild-type repressors bind to the operators, thereby preventing the expression of the lac structural genes. It occurs mainly in the absence of the inducer. Consider another example of a partial diploid having two sets of genes. The allolactose does not allow the binding of the repressor to the operator. Hence, the lac structural genes get expressed.
     

     Image 3: Lac I mutations

·        LacI^s (super-repressor mutations): These mutant genes show a dominant over the wild-type ones. Consider the partial diploid having two sets of genes one having LacI⁺ LacO⁺  LacZ^- LacY⁺ LacA⁺. The other having Lac I^- LacO⁺ LacZ⁺ Lac Y^- LacA⁺ genes. The repressor molecules synthesized by the lacI^- dominates over the wild-type ones. Hence, the lac structural genes do get expressed.

Positive control of lac operon:

It occurs only in the presence of lactose and absence of glucose. A protein known as catabolite activator protein (CAP) binds to the cyclic AMP (cAMP). It helps in positive regulation. The upstream to the promoter region involves a CAP site. The CAP-cAMP complex binds to the CAP site and helps in the binding of the RNA polymerase. Hence, it leads to the expression of genes. In the presence of glucose, the above complex cannot form. Hence, the lac structural genes do not get expressed. 

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