PRokaryotic gene regulation
We know that:
DNA is organized into genes
Genes store genetic information
This information is expressed trough transcription and translation
BUT the Q is… How is gene expression regulated???
Not all genes are expressed at all times in all situations in all the cells………
Cells of pancreas do not synthesize retinal pigment, and retinal cells do not make insulin.
Expression of a gene at the wrong time, or in the wrong place, or in abnormal amounts can lead to abnormal phenotype - cancer or cell death, even when the gene itself is normal.
Bacteria respond metabolically to the changes in their environment…
But they also regulate gene expression in order to synthesize products needed for different normal cellular activities (including DNA replication, recombination, repair, cell division)
In prokaryotes the regulation of gene expression mostly takes place at the level of transcription…
Ex: When lactose is present in growth medium of yeast, the organism synthesizes enzymes required for lactose metabolism. When lactose is absent, the enzymes are not produced.
Inducible enzymes – are produced only when specific chemical substrates are present.
Constitutive enzymes – that are produced continuously, regardless of the chemical makeup of the environment.
Sometimes the presence of a specific molecule inhibits the gene expression.
Ex: When enough amounts of tryptophan (trp) is present in cell’s environment it blocks the synthesis of enzymes, necessary for tryptophan production.
Thus, tryptophan represses transcription of genes that encode appropriate enzymes.
The system controlling tryptophane expression is a repressible system.
Whether a system is inducible or repressible, regulation may be under positive or negative control.
Negative control – is when gene expression is “shut off” by some form of regulatory molecule.
Positive control – is when transcription is “turned on” only if a regulatory molecule directly stimulates RNA production.
Lactose metabolism in E. coli is regulated by an inducible system
When lactose present – gene expression is induced.
When lactose is absent – gene expression is repressed.
In the presence of lactose the concentration of enzymes responsible for lactose metabolism increase rapidly (from few molecules to thousands per cell).
Thus, lactose serves as the inducer and the enzymes responsible for lactose metabolism are inducible.
In prokaryotes, the genes that code for enzymes with related functions (in this case lactose) are located in clusters and the transcription of these genes are often regulated by a single transcription regulatory region.
Operon – is a group of genes regulated and expressed together as a unit!
The location of this regulatory region is almost always upstream the gene cluster it controls – cis-acting site
cis-acting regulatory regions bind to the molecules that control transcription of the gene cluster, or to trans-acting molecules.
Binding of trans-acting molecules to the cis-acting site can regulate the expression of the gene cluster either negatively (by turning off transcription) or positively (by turning on transcription of genes in the cluster).
Structural and regulatory genes
Structural genes code for proteins needed for the normal functioning of the cell.
Regulator genes code for proteins that regulate other genes.
There are three structural genes in lac operon: Z, Y, A.
Lactose (lac operon) consists of three genes and an adjacent regulatory region.
The entire gene cluster works as one system to respond on the presence or absence of lactose.
The lacZ gene encodes b-galactosidase, an enzyme that converts disaccharide lactose to the monosaccharide glucose and galactose.
The lacY gene enzyme permease, an enzyme that facilitates the entry of lactose into the cell.
The lacA gene codes for enzyme transacetylase, which removes toxic by-products of lactose metabolism.
All three genes are closely linked to each other in the order of Z-Y-A.
They are transcribed as a single unit, resulting in a polysictronic mRNA.
This results in a coordinated regulation of all three genes, because a singe mRNA is translated into three gene products.
Lac operon: Negative Control
Repressor lacI gene regulates the transcription of structural genes by producing a repressor molecule.
Repressor molecule is allosteric - it reversibly interacts with another molecule and causes conformational change in the repressor’s shape and in its chemical activity.
Repressor normally binds to the DNA sequence of the operator region and inhibits the action of RNA polymerase and represses the transcription of the structural genes.
However, when lactose is present, the sugar binds to the repressor molecule and causes allosteic conformational change. Thus, repressor can not interact with operator DNA.
In the absence of repressor-operator interaction, RNA polymerase transcribes the structural genes and the enzymes necessary for lactose metabolism are produced. ☺
Because transcription occurs only when the repressor fails to bind to the operator region, regulation is said to be under negative control.
THUS:
In the absence of lactose, the enzymes encoded by the genes are not needed, and expression of genes encoding these enzymes is repressed.
When lactose is present, it indirectly promotes the transcription of the structural genes by interacting with the repressor.
If all lactose is metabolized nothing will be left to interact with the repressor. Repressor will be free and will interact with the operator DNA and transcription will be repressed.
Lac operon: positive control - catabolite-activating protein (CAP)
Lactose is cleaved into glucose and galactose by b-galactosidase.
Galactose must be converted to glucose in order to be used by the cell.
What if the cell is found in the environment, that is rich in lactose and glucose?
Glucose is the major carbon source for E. coli
It would not be energetically efficient for a cell to start transcription of the lac operon, make b-galactosidase and metabolize lactose, because what it really needs – glucose – is already present there.
A molecule catabolite-activating protein (CAP) helps activate expression of lac operon, but is able to inhibit expression when glucose is present.
This inhibition is called catabolic repression.
Transcription is initiated when RNA polymerase binds to the promoter region (found unstream 5’ from the coding sequences).
In lac operon, the promoter is located between the repressor (I) gene and the operator (O) region.
RNA polymerase binding is never efficient if CAP is not present to facilitate the process.
In the absence of glucose, CAP has positive control by binding to the CAP site, promoting RNA polymerase binding at the promoter and thus transcription.
For maximal transcription of the structural genes, the repressor must be bound by lactose and CAP must be bound to the CAP-binding site.
But what role does glucose play in inhibiting CAP binding when it is present?
A molecule cyclic-adenosine monophosphate (cAMP) binds to the CAP and only after this CAP is able to bind to the promoter.
The level of cAMP depends on the another enzyme – adenyl cyclase (which catalyses the conversion of ATP to cAMP).
The role of glucose is to catabolite the action of adenyl cyclase causing decrease in cAMP levels.
Under these conditions, CAP can not form the Cap-cAMP complex, which is essential to the positive control of transcription of the lac operon.
Alone, neither cAMP-CAP nor RNA polymerase has a strong tendency to bind to lac promoter DNA.
However, when both are together in the presence of lac promoter DNA, a tightly bound promoter region is formed. This is an example of cooperative binding.☺
The action of cAMP-CAP makes positive regulation.
Regulation of the lac operon by catabolite repression results in efficient energy use, because the presence of glucose will prevent the need for the metabolism of lactose, should the lactose also be available to the cell.
Thus, a combination of positive and negative regulatory mechanisms determine transcription levels of the lac operon.
The tryptophan (trp) operon in E. coli is a repressible gene system
If tryptophan is present in sufficient amounts in the growth medium, the enzymes necessary for it synthesis are not produced.
It is energetically beneficiary for bacteria to repress expression of genes involved in tryptophan synthesis when enough amounts of tryptophan is present in the growth medium.
Tryptophan synthesis involves several enzymes, synthesized by five genes, located adjacent to each other on E. coli chromosome.
In the presence of tryptophan all genes are repressed and non of the enzymes are produced.
The trp operon model suggest the existence of normally inactive repressor, that alone can not interact with the operator.
But repressor can bind to allosteric tryptophan and this complex binds then to the operator and represses transcription.
Thus, when tryptophan is present, the system is repressed and the enzymes are not made.
This repressible system is under negative control, because this regulatory complex inhibits the transcription of the operon.