1. Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 1. The logic of prokaryotic transcriptional regulation In addition to the sigma factors that allow RNA polymerase to bind the promoter, another type of DNA-protein interaction regulates whether or not promoter-driven transcription occurs. In addition to the sigma factors that allow RNA polymerase to bind the promoter, another type of DNA-protein interaction regulates whether or not promoter-driven transcription occurs. DNA segments near the promoter serve as protein-binding sites for regulatory proteins called activators and repressors; these sites on DNA are termed operators. DNA segments near the promoter serve as protein-binding sites for regulatory proteins called activators and repressors; these sites on DNA are termed operators. For some genes, the binding of an activator protein to its target DNA site is a necessary prerequisite for transcription to begin (positive regulation). For other genes, preventing the binding of a repressor protein to its target site is a necessary prerequisite for transcription to begin (negative regulation).

2. Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 2. Activators, repressors, effectors For activator or repressor proteins to do their job, each must be able to exist in two states: one that can bind its DNA targets and one that cannot. The binding state must be in accord with the cellular environment; that is, be appropriate for a given set of physiological conditions. A site on the regulator protein interacts with small molecules called allosteric effectors; these act as toggle switches that sets the DNA-binding domain in one of two modes: functional or nonfunctional. An allosteric effector binds to the allosteric site of the regulatory protein in such a way that it changes the structure of the DNA-binding domain. Some activator or repressor proteins must bind to their allosteric effectors to bind DNA. Others can bind DNA only in the absence of their allosteric effectors.

3. Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 3. A molecular switch DNA-bound activator proteins act at the level of transcription initiation, by physically helping to bind RNA polymerase to its nearby promoter. A DNA-bound repressor protein typically acts either by physically interfering with the binding of RNA polymerase to its promoter (blocking transcription initiation) or by impeding the movement of RNA polymerase along the DNA chain (blocking transcription elongation).

4. Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 4. Regulation of the Lactose System A relevant example of transcriptional regulation in prokaryotes is the control of the enzymes necessary for lactose metabolism in E. coli. Most of the models and mechanisms involved in this specific system have been revealed by genetic analyses of mutated bacterial strains. A relevant example of transcriptional regulation in prokaryotes is the control of the enzymes necessary for lactose metabolism in E. coli. Most of the models and mechanisms involved in this specific system have been revealed by genetic analyses of mutated bacterial strains.  Presumably because of energy-efficiency considerations, two environmental conditions have to be satisfied for the lactose metabolic enzymes to be expressed.  One condition is that lactose must be present in the environment. It would be inefficient for the cell to produce the lactose metabolic enzymes in circumstances where there is no substrate to metabolize.  The other condition is that glucose should not be present in the cell's environment. Because glucose metabolism yields more usable energy to the cell than does lactose metabolism, mechanisms have evolved that prevent the synthesis of the enzymes for lactose metabolism in the presence of glucose.

5. Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 5. Introducing the operon A simplified lac operon model. The three genes Z, Y, and A are coordinately expressed. The product of the I gene, the repressor, blocks the expression of the Z, Y, and A genes by interacting with the operator (O). The inducer can inactivate the repressor, thereby preventing interaction with the operator. When this happens, the operon is fully expressed.

6. Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 6. The metabolism of lactose The metabolism of lactose requires two enzymes: a permease to transport lactose into the cell and b-galactosidase to cleave the lactose molecule to yield glucose and galactose (Figure 14-4). Permease and b-galactosidase are encoded by two contiguous genes, Z and Y, respectively. A third gene, the A gene, encodes an additional enzyme, termed transacetylase, but this enzyme is not required for lactose metabolism. All three genes are transcribed into a single, multigenic messenger RNA (mRNA) molecule. Regulation of the production of this mRNA coordinates the regulation of the synthesis of all three enzymes.

7. Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 7. The gene for the Lac repressor and the lac operator site A fourth gene, the I gene, encodes the Lac repressor protein, so named because it can block the expression of the Z, Y, and A genes. The I gene happens to map fairly near the Z, Y, and A genes, but this proximity does not seem to be important to its function. A fourth gene, the I gene, encodes the Lac repressor protein, so named because it can block the expression of the Z, Y, and A genes. The I gene happens to map fairly near the Z, Y, and A genes, but this proximity does not seem to be important to its function. The operator (O) is the site on the DNA to which the Lac repressor binds. It is located between the promoter and the Z gene near the point at which transcription of the multigenic mRNA begins. The operator (O) is the site on the DNA to which the Lac repressor binds. It is located between the promoter and the Z gene near the point at which transcription of the multigenic mRNA begins.

8. Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 8. The lac operon is regulated by lactose The P, O, Z, Y, and A segments constitute an operon, which is a genetic unit of coordinate expression. The interaction between the lac operator site on the DNA and the Lac repressor is crucial to proper regulation of the lac operon. The Lac repressor is a molecule with two recognition sites, a DNA-binding site that can recognize the specific operator DNA sequence for the lac operon and an allosteric site that binds the lactose allosteric effector and similar molecules (analogs of lactose).

9. Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 9. The lac repressor Lac repressor is a tetrameric protein organized as a dimer of dimers. Each component homodimer forms one DNA binding region from two equivalent chains. The lac operator sequence is an almost perfect palyndrome, recognized by inserting one helix-turn-helix motif from each chain of the lac repressor dimer into the DNA major groove of the half palyndrome. This helix-turn-helix motif is found to be common to a variety of bacterial and phage repressor or DNA binding proteins

10. Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 10. The complex between the lac repressor and the lac operator The DNA-binding site of the Lac repressor is able to bind with high affinity to only one DNA sequence in the entire E. coli genome, the lac operator. The specificity of high- affinity DNA binding ensures that the repressor will bind only to the site on the DNA near the genes that it is controlling and not to random sites distributed throughout the chromosome. By binding to the operator, the repressor prevents transcription by RNA polymerase that has bound to its lac promoter site

11. Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 11. A second mechanism of control In the absence of lactose, the Lac repressor binds to the lac operator site and prevents transcription of the lac operon, by blocking the progression of RNA polymerase transcription. In the absence of lactose, the Lac repressor binds to the lac operator site and prevents transcription of the lac operon, by blocking the progression of RNA polymerase transcription. Consequently, all of the structural genes of the lac operon (the Z, Y, and A genes) are repressed, and there is no b-galactosidase, b-galactoside permease, or transacetylase in the cell. Consequently, all of the structural genes of the lac operon (the Z, Y, and A genes) are repressed, and there is no b-galactosidase, b-galactoside permease, or transacetylase in the cell. In contrast, when lactose is present, it binds to the allosteric site of the Lac repressor, thereby inactivating the operator DNA-binding site of the Lac repressor protein. This inactivation permits the induction of transcription of the structural genes of the lac operon and, through the translation of the multigenic mRNA, the enzymes b- galactosidase, b-galactoside permease, and transacetylase now appear in the cell in a coordinated fashion In contrast, when lactose is present, it binds to the allosteric site of the Lac repressor, thereby inactivating the operator DNA-binding site of the Lac repressor protein. This inactivation permits the induction of transcription of the structural genes of the lac operon and, through the translation of the multigenic mRNA, the enzymes b- galactosidase, b-galactoside permease, and transacetylase now appear in the cell in a coordinated fashion However, there is more to the regulation of lac operon transcription. The above mechanism satisfy only one of the conditions that the lac operon should obey; the entire system also requires a second environmental condition, namely, that glucose is not present in the environment of the cell. However, there is more to the regulation of lac operon transcription. The above mechanism satisfy only one of the conditions that the lac operon should obey; the entire system also requires a second environmental condition, namely, that glucose is not present in the environment of the cell.

12. Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 12. The effect of glucose An additional control system is superimposed on the repressor operator system. This control system is thought to have evolved because the cell can capture more energy from the breakdown of glucose than it can from the breakdown of other sugars. An additional control system is superimposed on the repressor operator system. This control system is thought to have evolved because the cell can capture more energy from the breakdown of glucose than it can from the breakdown of other sugars. If both lactose and glucose are present, the synthesis of b- galactosidase is not induced until all the glucose has been utilized. If both lactose and glucose are present, the synthesis of b- galactosidase is not induced until all the glucose has been utilized. Thus, the cell conserves its energy pool used, for example, to synthesize the Lac enzymes by utilizing any existing glucose before going through the energy-expensive process of creating new machinery to metabolize lactose. Thus, the cell conserves its energy pool used, for example, to synthesize the Lac enzymes by utilizing any existing glucose before going through the energy-expensive process of creating new machinery to metabolize lactose. It is a breakdown product of glucose (the identity of this catabolite is as yet unknown) that prevents activation of the lac operon by lactose; this is the catabolite repression mechanism. It is a breakdown product of glucose (the identity of this catabolite is as yet unknown) that prevents activation of the lac operon by lactose; this is the catabolite repression mechanism.

13. Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 13. cAMP and CAP A glucose catabolite modulates the level of an important cellular constituent, cyclic adenosine monophosphate (cAMP). A glucose catabolite modulates the level of an important cellular constituent, cyclic adenosine monophosphate (cAMP). When glucose is present in high concentrations, the cell's cAMP concentration is low; as the glucose concentration decreases, the cellular concentration of cAMP increases correspondingly. When glucose is present in high concentrations, the cell's cAMP concentration is low; as the glucose concentration decreases, the cellular concentration of cAMP increases correspondingly. The high concentration of cAMP is necessary for activation of the lac operon. The high concentration of cAMP is necessary for activation of the lac operon. cAMP is an effector of a protein, called CAP (catabolite activator protein), which is coded by the crp gene. In absence of cAMP, CAP cannot bind to the CAP site, while bound to cAMP, CAP is able to bind to the CAP site. cAMP is an effector of a protein, called CAP (catabolite activator protein), which is coded by the crp gene. In absence of cAMP, CAP cannot bind to the CAP site, while bound to cAMP, CAP is able to bind to the CAP site. The DNA-bound CAP is then able to interact physically with RNA polymerase and essentially increase the affinity of RNA polymerase for the lac promoter. CAP is an activator protein. The DNA-bound CAP is then able to interact physically with RNA polymerase and essentially increase the affinity of RNA polymerase for the lac promoter. CAP is an activator protein.

14. Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 14. CAP binding to DNA-CAP binding site 11-12 The CAP protein active sites are modified by the presence of cAMP so that the complex binds at the proper DNA site in the promoter of the lac operon. 11-12

15. Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 15. The 5’ lac operator control region

16. Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 16. The effect of cAMP-ligated CAP

17. Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 17. A summary of the lac operon control