Genetica per Scienze Naturali a.a. 05-06 prof S. Presciuttini 1. The logic of prokaryotic transcriptional regulation In addition to the sigma factors that.

Genetica per Scienze Naturalia.a. 05-06 prof S. Presciuttini

1. The logic of prokaryotic transcriptional regulation In addition to the sigma factors that allow RNA polymerase to bind In addition to the sigma factors that allow RNA polymerase to bind

the promoter, anothe promoter, another type of DNAther type of DNA--protein interaction regulates protein interaction regulates whether or not promoter-driven transcription occurs.whether or not promoter-driven transcription occurs.

DNA segments near the promoter serve as protein-binding sitesDNA segments near the promoter serve as protein-binding sites for for regulatory proteinsregulatory proteins called called activatorsactivators and and repressorsrepressors;; these sites these sites on on DNA DNA are termed are termed operatorsoperators..

For some genes, the For some genes, the bindingbinding of an activator protein to its of an activator protein to its target DNA site is a necessary target DNA site is a necessary prerequisite for transcription prerequisite for transcription to beginto begin ( (positive regulationpositive regulation). ). For other genes, For other genes, preventing preventing the bindingthe binding of a repressor of a repressor protein to its target site is a protein to its target site is a necessary prerequisite for necessary prerequisite for transcription to begintranscription to begin ((negative regulationnegative regulation).).


2. Activators, repressors, effectors

For activator or repressor proteins to do their job, each must be able to 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. 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 The binding state must be in accord with the cellular environment; that is, be appropriate for a given set of physiological conditions.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. A molecular switch

DNA-bound activator proteinDNA-bound activator proteinss act at act at the level of transcription initiation, the level of transcription initiation, by physically helping to by physically helping to bindbind RNA RNA polymerase to its nearby promoter. A polymerase to its nearby promoter. A DNA-bound repressor protein DNA-bound repressor protein typically acts either by physically typically acts either by physically interfering with the binding of RNA interfering with the binding of RNA polymerase to its promoter (blocking polymerase to its promoter (blocking transcription initiation) or by transcription initiation) or by impeding the movement of RNA impeding the movement of RNA polymerase along the DNA chain polymerase along the DNA chain (blocking transcription elongation).(blocking transcription elongation).


4. Regulation of the Lactose System A relevant example ofA relevant example of transcriptional regulation in prokaryotes transcriptional regulation in prokaryotes is the is the

controlcontrol of the enzymes necessary for lactose metabolism in E of the enzymes necessary for lactose metabolism in E.. coli. coli. Most of the models and mechanisms involved in this specific system Most of the models and mechanisms involved in this specific system have been revealed by genetic analyses of mutated bacterial strains.have been revealed by genetic analyses of mutated bacterial strains. Presumably because of energy-efficiency considerations, two environmental Presumably because of energy-efficiency considerations, two environmental

conditions have to be satisfied for the lactose metabolic enzymes to be conditions have to be satisfied for the lactose metabolic enzymes to be expressed.expressed.

One condition is that lactose must be present in the environment. One condition is that lactose must be present in the environment. IIt would be t would be inefficient for the cell to produce the lactose metabolic enzymes in inefficient for the cell to produce the lactose metabolic enzymes in circumstances where there is no substrate to metabolize.circumstances where there is no substrate to metabolize.

The other condition is that glucose The other condition is that glucose should notshould not be present in the cell's be present in the cell's environment. Because glucose metabolism yields more usable energy to the cell environment. Because glucose metabolism yields more usable energy to the cell than does lactose metabolism, mechanisms have evolved that prevent the than does lactose metabolism, mechanisms have evolved that prevent the

synthesis of the enzymes for lactose metabolism in the presence of glucose.synthesis of the enzymes for lactose metabolism in the presence of glucose.


5. Introducing the operonA simplified lac operon model. The three genes Z, Y, and A are A simplified lac operon model. The three genes Z, Y, and A are coordinately expressed. The product of the I gene, the repressor, blocks 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 the expression of the Z, Y, and A genes by interacting with the operator (O). The inducer can inactivate the repressor, thereby preventing (O). The inducer can inactivate the repressor, thereby preventing interaction with the operator. When this happens, the operon is fully interaction with the operator. When this happens, the operon is fully expressed. expressed.


6. The metabolism of lactose

The metabolism of lactose requires two enzymes: a permease to transport lactose into 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 the cell and b-galactosidase to cleave the lactose molecule to yield glucose and galactose. Permease and b-galactosidase are encoded by two contiguous genes, Z and galactose. 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 Y, respectively. A third gene, the A gene, encodes an additional enzyme, termed transacetylase, but this enzyme is not required for lactose metabolism. transacetylase, but this enzyme is not required for lactose metabolism.

All three genes are transcribed All three genes are transcribed into a single, multigenic into a single, multigenic messenger RNA (mRNA) messenger RNA (mRNA) molecule. Regulation of the molecule. Regulation of the production of this mRNA production of this mRNA coordinates the regulation of the coordinates the regulation of the synthesis of all three enzymes.synthesis of all three enzymes.


7. The gene for the Lac repressorand the lac operator site

A fourth gene, the I gene, encodes the Lac repressor protein, A fourth gene, the I gene, encodes the Lac repressor protein, so named because it can block the expression of the Z, Y, 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, 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 Y, and A genes, but this proximity does not seem to be important to its function.important to its function.

The operator (O) is the site on the DNA to which the Lac The operator (O) is the site on the DNA to which the Lac repressor binds. It is located between the promoter and the Z repressor binds. It is located between the promoter and the Z gene near the point at which transcription of the multigenic gene near the point at which transcription of the multigenic mRNA begins.mRNA begins.


8. The lac operon is regulated by lactoseThe P, O, Z, Y, and A segments constitute an operon, which is a genetic unit of 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 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 the Lac repressor is crucial to proper regulation of the lac operon. The Lac repressor is a molecule with two recognition sitesa molecule with two recognition sites, , a DNA-binding site that can recognize the a DNA-binding site that can recognize the specific operator DNA sequence for the lac operon and an allosteric site that binds 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).lactose allosteric effector and similar molecules (analogs of lactose).


9. The Lac repressorLac repressor is a tetrameric protein organized as a dimer of dimers. Lac repressor is a tetrameric protein organized as a dimer of dimers. Each component homodimer forms one DNA binding region from two Each component homodimer forms one DNA binding region from two equivalent chains. The lac operator sequence equivalent chains. The lac operator sequence is an almost perfect is an almost perfect palyndromepalyndrome, recognized by inserting one helix-turn-helix motif from , recognized by inserting one helix-turn-helix motif from each chain of the lac repressor dimer into the DNA major groove of the each chain of the lac repressor dimer into the DNA major groove of the half half palyndromepalyndrome. .

This helix-turn-helix motif is found to be This helix-turn-helix motif is found to be common to a variety of bacterial and phage common to a variety of bacterial and phage repressor or DNA binding proteinsrepressor or DNA binding proteins


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 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 genomeone DNA sequence in the entire E. coli genome, , the lac operator. The specificity of the lac operator. The specificity of high-affinity DNA binding ensures that the repressor will bind only to the site on the 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 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 the chromosome. By binding to the operator, the repressor prevents transcription by RNA polymerase that has bound to its lac promoter siteRNA polymerase that has bound to its lac promoter site


11. A second mechanism of control In the absence In the absence of of lactose, the Lac repressor binds to the lac operator site and lactose, the Lac repressor binds to the lac operator site and

prevents transcription of the lac operonprevents transcription of the lac operon, by, by block blockinging the progression of RNA the progression of RNA polymerase transcription.polymerase transcription.

Consequently, all of the structural genes of the lac operon (the Z, Y, and A genes) 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 are repressed, and there is no b-galactosidase, b-galactoside permease, or transacetylase in the cell.transacetylase in the cell.

In contrast, when lactose is present, it binds to the allosteric site of the Lac 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 repressor, thereby inactivating the operator DNA-binding site of the Lac repressor protein. This inactivation permits the induction of transcription of the structural 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 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 enzymes b-galactosidase, b-galactoside permease, and transacetylase now appear in the cell in a coordinated fashionthe cell in a coordinated fashion

HHowever, there is more to the regulation of lac operon transcription. owever, there is more to the regulation of lac operon transcription. The above The above mechanism satisfy only one of the conditionsmechanism satisfy only one of the conditions that that the lac operon the lac operon should obey; should obey; the the entire systementire system also requires a second environmental condition also requires a second environmental condition, , namely, that glucose namely, that glucose is not present in the environment of the cell. is not present in the environment of the cell.


12. The effect of glucose An additional control system is superimposed on the repressorAn additional control system is superimposed on the repressor operator operator

system. This control system is thought to have evolved because the cell can 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 capture more energy from the breakdown of glucose than it can from the breakdown of other sugars.breakdown of other sugars.

If both lactose and glucose are present, the synthesis of b-galactosidase is not If both lactose and glucose are present, the synthesis of b-galactosidase is not induced until all the glucose has been utilized.induced until all the glucose has been utilized.

Thus, the cell conserves its energy pool used, for example, to synthesize the Thus, the cell conserves its energy pool used, for example, to synthesize the Lac enzymes by utilizing any existing glucose before going through the Lac enzymes by utilizing any existing glucose before going through the energy-expensive process of creating new machinery to metabolize lactoseenergy-expensive process of creating new machinery to metabolize lactose . .

It isIt is a breakdown product of glucose (the identity of this catabolite is as yet a breakdown product of glucose (the identity of this catabolite is as yet unknown) unknown) that that prevents activation of the lac operon by lactoseprevents activation of the lac operon by lactose; this is ; this is the the catabolite repression catabolite repression mechanismmechanism..


13. cAMP and CAP AA glucose catabolite modulates the level of an important cellular glucose catabolite modulates the level of an important cellular

constituentconstituent, , cyclic adenosine monophosphate (cAMP).cyclic adenosine monophosphate (cAMP). When glucose is present in high concentrations, the cell's cAMP When glucose is present in high concentrations, the cell's cAMP

concentration is low; as the glucose concentration decreases, the concentration is low; as the glucose concentration decreases, the cellular concentration of cAMP increases correspondingly.cellular concentration of cAMP increases correspondingly.

The high concentration of cAMP is necessary for activation of the lac The high concentration of cAMP is necessary for activation of the lac operon.operon.

cAMP cAMP is an effector of a is an effector of a protein, called CAP (catabolite activator protein, called CAP (catabolite activator protein), protein), which is codedwhich is coded by the by the crpcrp gene. gene. In absence of In absence of cAMP , CAP cAMP , CAP cannot bind to the CAP sitecannot bind to the CAP site, while, while bound tobound to cAMP, CAP is able to cAMP, CAP is able to bind to the CAP site.bind to the CAP site.

The DNA-bound CAP is then able to interact physically with RNA The DNA-bound CAP is then able to interact physically with RNA polymerase and essentially increase the affinity of RNA polymerase polymerase and essentially increase the affinity of RNA polymerase for the lac promoter. for the lac promoter. CAP is an activator protein.CAP is an activator protein.


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. The 5’ lac operator control region


16. The effect of cAMP-ligated CAP


17. A summary of the lac operon control

Genetica per Scienze Naturali a.a. 05-06 prof S. Presciuttini 1. The logic of prokaryotic transcriptional regulation In addition to the sigma factors that.

Documents

dnabinding domain

dnabound repressor protein

lactose metabolism

dna chain

type of dna

dna targets

target dna site

proteinbinding sites