Regulation of protein synthesis in Prokaryotes

Prokaryotes are unicellular organisms that lack a nucleus and other membrane-bound organelles. They have a simple and compact genome that consists of a single circular chromosome and sometimes plasmids. Prokaryotes need to adapt quickly to changes in their environment, such as the availability of nutrients, in order to survive and grow. One of the ways they do this is by regulating the synthesis of proteins, which are the main functional molecules in the cell.

Protein synthesis is the process of translating the genetic information encoded in mRNA into amino acid sequences that form polypeptides. Protein synthesis requires a lot of energy and resources, so prokaryotes need to balance the production of proteins with the demand and supply of nutrients. If a nutrient is abundant, prokaryotes can synthesize more proteins that are involved in its metabolism or utilization. If a nutrient is scarce, prokaryotes can reduce or stop the synthesis of proteins that are not essential or that consume the nutrient.

Regulation of protein synthesis in prokaryotes occurs mainly at the transcriptional level, and involves genetic units known as operons. An operon is a set of genes that are adjacent to one another in the genome and are coordinately controlled; that is, the genes are either all turned on or all turned off. Operons contain promoter regions where proteins bind and facilitate or inhibit the binding of RNA polymerase. When RNA polymerase transcribes the structural genes of an operon, a polycistronic mRNA (i.e., an mRNA that codes for more than one polypeptide) is produced.

There are different mechanisms by which prokaryotes regulate protein synthesis based on nutrient supply, such as induction, repression, positive control, catabolite repression, and attenuation. These mechanisms involve the interaction of small molecules (such as sugars or amino acids) with regulatory proteins (such as repressors or activators) that modulate the transcription of operons. In this article, we will discuss these mechanisms and provide examples of how prokaryotes respond to changes in glucose and amino acid availability.

E. coli is a common prokaryote that can use different sugars as energy sources, depending on their availability in the environment. Glucose is the preferred sugar for E. coli, as it can be easily metabolized through glycolysis and the citric acid cycle. However, if glucose is scarce or absent, E. coli can switch to other sugars, such as lactose, arabinose, or galactose.

To do so, E. coli needs to regulate the expression of different operons that encode the enzymes and transporters for each sugar. For instance, the lac operon contains genes for lactose uptake and breakdown, while the ara operon contains genes for arabinose utilization. These operons are controlled by various mechanisms that sense the presence or absence of glucose and the alternative sugars.

One of these mechanisms is catabolite repression, which involves a protein called catabolite activator protein (CAP) and a small molecule called cyclic AMP (cAMP). When glucose levels are high, cAMP levels are low, and CAP cannot bind to the DNA. This prevents the transcription of operons that are activated by CAP, such as lac and ara. When glucose levels are low, cAMP levels rise, and CAP binds to cAMP and to the DNA. This facilitates the transcription of operons that are activated by CAP, but only if the corresponding sugar is present.

Another mechanism is induction, which involves a protein called lac repressor and a molecule called allolactose. When lactose is absent, the lac repressor binds to the operator region of the lac operon and blocks its transcription. When lactose is present, it is converted into allolactose by an enzyme called beta-galactosidase. Allolactose binds to the lac repressor and changes its shape, making it unable to bind to the operator. This allows the transcription of the lac operon and the production of enzymes for lactose metabolism.

By using these mechanisms, E. coli can adjust its metabolism to the availability of glucose and other sugars in its environment. This allows it to obtain or conserve energy most efficiently . However, chronic exposure to glucose can also have negative effects on E. coli`s growth and health, such as increased production of harmful molecules called advanced glycation end products (AGEs). These effects can be reduced by preventing E. coli from processing excess glucose or by adding compounds that inhibit AGE formation.

An operon is a set of genes that are adjacent to one another in the genome and are coordinately controlled; that is, the genes are either all turned on or all turned off. An operon is made up of several structural genes arranged under a common promoter and regulated by a common operator. The operator controls the activity of the structural genes by interacting with a regulator gene. The structural genes have related functions and code for proteins that are involved in a common metabolic pathway.

Operons allow prokaryotes to regulate the expression of multiple genes in response to environmental changes or nutritional needs. By grouping genes together, operons reduce the amount of DNA and RNA needed for gene expression and ensure that the genes are expressed in a coordinated manner. Operons also enable feedback mechanisms that can turn off or turn on gene expression depending on the presence or absence of certain molecules.

There are different types of operons that differ in their modes of regulation. Some operons are inducible, meaning that they are normally off but can be turned on by an inducer molecule. Some operons are repressible, meaning that they are normally on but can be turned off by a corepressor molecule. Some operons are under positive control, meaning that they require an activator protein to enhance transcription. Some operons are under negative control, meaning that they are inhibited by a repressor protein that binds to the operator. Some operons are subject to catabolite repression, meaning that they are repressed by glucose or other preferred carbon sources. Some operons are regulated by attenuation, meaning that they use a mechanism that involves premature termination of transcription.

In the following sections, we will discuss some examples of operons and how they regulate protein synthesis in prokaryotes.

: Question answering result from Operon - Wikipedia : Regulation of Protein Synthesis on the Basis of Nutrient Supply : Regulation of Protein Synthesis on the Basis of Nutrient Supply : Regulation of Protein Synthesis on the Basis of Nutrient Supply

Induction is the process whereby an inducer (a small molecule) stimulates the transcription of an operon. The inducer is frequently a sugar (or a metabolite of the sugar), and the proteins produced from the inducible operon allow the sugar to be metabolized .

The working mechanism of induction involves a repressor protein that binds to the operator sequence of the operon and blocks the access of RNA polymerase to the promoter. When the inducer is present, it binds to the repressor and changes its conformation, making it unable to bind to the operator. This allows RNA polymerase to bind to the promoter and transcribe the operon .

The following code block shows a simplified diagram of induction:

Inducer + Repressor -> Inducer-Repressor complex
Inducer-Repressor complex cannot bind to Operator
RNA polymerase can bind to Promoter and transcribe Operon

Induction is a type of positive control, because it enhances the transcription of an operon. Induction allows the cell to respond to changes in nutrient availability and produce only the enzymes that are needed for a specific metabolic pathway .

The Lac operon is a set of genes that are involved in the transport and metabolism of lactose in E. coli and other bacteria . The Lac operon consists of three structural genes: lacZ, lacY, and lacA. lacZ encodes an enzyme called β-galactosidase that cleaves lactose into glucose and galactose. lacY encodes a membrane protein called β-galactoside permease that transports lactose into the cell. lacA encodes an enzyme called β-galactoside transacetylase that modifies some β-galactosides.

The Lac operon is considered an inducible operon because it is usually turned off (repressed), but can be turned on (induced) in the presence of lactose. The induction of the Lac operon is controlled by two regulatory proteins: the lac repressor and the catabolite activator protein (CAP).

The lac repressor is a protein that binds to a DNA sequence called the operator, which is located between the promoter and the structural genes of the Lac operon. When the lac repressor binds to the operator, it blocks the access of RNA polymerase to the promoter, and thus prevents the transcription of the Lac operon.

The lac repressor is sensitive to the presence of lactose, or more precisely, to a derivative of lactose called allolactose. Allolactose is formed by the action of β-galactosidase on a small fraction of lactose molecules. Allolactose acts as an inducer for the Lac operon, meaning that it binds to the lac repressor and changes its shape, making it unable to bind to the operator . This allows RNA polymerase to bind to the promoter and transcribe the Lac operon, leading to the production of more β-galactosidase, β-galactoside permease, and β-galactoside transacetylase.

The catabolite activator protein (CAP) is another protein that regulates the expression of the Lac operon. CAP binds to a DNA sequence called the CAP site, which is located upstream of the promoter of the Lac operon. When CAP binds to the CAP site, it enhances the binding of RNA polymerase to the promoter, and thus increases the transcription of the Lac operon.

However, CAP can only bind to DNA when it is complexed with a small molecule called cyclic AMP (cAMP). cAMP is produced from ATP by an enzyme called adenylate cyclase, and its level in the cell depends on the availability of glucose. When glucose is abundant, adenylate cyclase is inhibited, and cAMP levels are low. This means that CAP cannot bind to DNA, and thus cannot activate the Lac operon. When glucose is scarce, adenylate cyclase is active, and cAMP levels are high. This means that CAP can bind to DNA, and thus can activate the Lac operon.

Therefore, the induction of the Lac operon depends on both lactose and glucose levels in the cell. The Lac operon is only expressed when lactose is present (to inactivate the lac repressor) and glucose is absent (to activate CAP). This ensures that E. coli uses lactose as an alternative energy source only when glucose is not available.

Repression is the process whereby a corepressor (a small molecule) inhibits the transcription of an operon. The corepressor is usually an amino acid, and the proteins produced from the repressible operon are involved in the synthesis of the amino acid .

Working of a Repressor:

• The corepressor binds to the repressor, activating it .
• The active repressor binds to the operator .
• RNA polymerase, therefore, cannot bind to the promoter, and the operon is not transcribed .
• The cell stops producing the structural proteins encoded by the operon .

Repression allows the cell to conserve energy and resources by avoiding the synthesis of amino acids that are already present in the environment . Repressors are more common in prokaryotes than in eukaryotes.

The tryptophan operon is a group of five genes that encode enzymes for the synthesis of the amino acid tryptophan in E. coli bacteria. The expression of this operon is regulated by repression, which means that it is turned off when tryptophan levels are high in the cell.

The regulation by repression involves a repressor protein and a corepressor molecule. The repressor protein is encoded by a gene called trpR, which is located outside the operon and is constitutively expressed. The corepressor molecule is tryptophan itself, or a metabolite derived from it.

When tryptophan is scarce in the cell, the repressor protein is inactive and cannot bind to the operator sequence, which is a DNA segment between the promoter and the first gene of the operon. This allows RNA polymerase to bind to the promoter and transcribe the operon, producing a polycistronic mRNA that codes for the five enzymes needed for tryptophan synthesis.

When tryptophan is abundant in the cell, it binds to the repressor protein and changes its shape, activating it. The activated repressor-corepressor complex then binds to the operator sequence and blocks the access of RNA polymerase to the promoter. This prevents the transcription of the operon and stops the production of the enzymes.

The repression mechanism ensures that E. coli does not waste energy and resources by making more tryptophan than it needs. It also allows E. coli to respond quickly to changes in tryptophan availability by switching on or off the operon as needed.

The following diagram illustrates how repression works in the tryptophan operon:

trpR gene trp operon
| |
| | promoter operator trpE trpD trpC trpB trpA
| | | | | | | | |
| V V V V V V V V
| ---------------------------------------------------------------------->
| <--------------------------> mRNA
|
V
trp repressor
|
V
+-----------------+
| |
| +----------+ |
| | | |
| |trp repressor|<----->tryptophan (corepressor)
| | | |
| +----------+ |
| |
+-----------------+
|
V
trp repressor-corepressor complex
|
V
+-----------------+
| |
| +----------+ |
| | | |
| |trp repressor|<----->tryptophan (corepressor)
| | | |
| +----------+ |
| || |
+------||---------+
||
||
||
||
||
||
\/
trpR gene trp operon
| |
| | promoter operator trpE trpD trpC trpB trpA
| | | | | | | | |
V V V V V V V V V
---------------------------------------------------------------------->
X X<----------X
^ ^
| |
RNA polymerase blocked by repressor-corepressor complex

Positive control is a mechanism by which a small molecule or a protein activates the transcription of an operon. The activator protein binds to a specific site near the promoter and facilitates the binding of RNA polymerase to the promoter. The operon is then transcribed, and the proteins required for the metabolic process are produced .

Positive control often occurs in operons that are involved in catabolic pathways, such as the breakdown of sugars other than glucose. These operons are usually turned off when glucose is present, because glucose is the preferred energy source for bacteria. However, when glucose is scarce and another sugar is available, the operon can be turned on by an activator protein that senses the presence of the alternative sugar .

One example of positive control is the arabinose operon (ara), which encodes proteins that allow the bacteria to utilize arabinose as a carbon source. The ara operon has a unique regulatory protein called AraC, which can act as both a repressor and an activator depending on its conformation. When arabinose is absent, AraC binds to two sites on the DNA and forms a loop that prevents RNA polymerase from accessing the promoter. When arabinose is present, it binds to AraC and changes its shape, allowing it to bind to a different site near the promoter and recruit RNA polymerase. The ara operon is then transcribed, and the enzymes for arabinose metabolism are produced .

Positive control allows bacteria to adapt to changing environmental conditions and use alternative energy sources when glucose is not available. It also ensures that the enzymes for catabolic pathways are only produced when they are needed, saving energy and resources for the cell. Positive control is one of the many ways that bacteria regulate their gene expression in response to nutrient supply.

Catabolite repression is a global regulatory mechanism that allows bacteria to adapt quickly to a preferred (rapidly metabolizable) carbon and energy source first, and inhibits the expression and activities of functions for the use of secondary carbon sources when the preferred one is present. This allows bacteria to selectively use substrates from a mixture of different carbon sources.

The most common example of catabolite repression is the inhibition of the lac operon by glucose in Escherichia coli. Glucose causes catabolite repression by lowering the intracellular levels of cyclic AMP (cAMP), which is required for the activation of the lac operon by the catabolite activator protein (CAP) .

The mechanism by which glucose lowers cAMP levels involves the phosphotransferase system (PTS), which is responsible for the transport and phosphorylation of glucose and other sugars. The PTS consists of several enzymes, one of which is enzyme IIA (EIIA), which transfers a phosphate group from phosphoenolpyruvate (PEP) to incoming sugars. When glucose is abundant, EIIA is mostly unphosphorylated, and it inhibits the activity of adenylyl cyclase, the enzyme that produces cAMP from ATP. When glucose is scarce, EIIA is mostly phosphorylated, and it activates adenylyl cyclase, leading to high levels of cAMP .

When cAMP levels are high, cAMP binds to CAP and forms a complex that binds to a specific site near the promoter of the lac operon. This complex facilitates the binding of RNA polymerase to the promoter and enhances the transcription of the lac operon. However, this is not enough for the expression of the lac operon; lactose must also be present to remove the lac repressor from the operator sequence.

When cAMP levels are low, CAP cannot bind to the promoter site and RNA polymerase cannot bind efficiently to the promoter. The transcription of the lac operon is therefore repressed. This prevents the production of unnecessary enzymes for lactose metabolism when glucose is available.

Catabolite repression also affects other operons in E. coli and other bacteria, such as the arabinose (ara) operon, which requires both cAMP-CAP and arabinose for its expression. Catabolite repression can also be achieved by different mechanisms in different bacteria, such as translation repression by an RNA-binding protein in pseudomonads, or glucose kinase-mediated regulation in actinobacteria. Catabolite repression has a key role in the expression of virulence factors in many pathogenic bacteria, and it is a promising target for antimicrobial therapy.

The lac operon is an example of an operon that is subject to both induction and catabolite repression. Induction occurs when lactose is present and binds to the lac repressor, preventing it from blocking transcription. Catabolite repression occurs when glucose is present and inhibits the production of cAMP, which is required for the activation of transcription by CAP.

When both glucose and lactose are present in the medium, E. coli prefers to use glucose as its main carbon and energy source. In this case, the lac operon is repressed by two mechanisms: (1) the lac repressor binds to the operator and blocks RNA polymerase from transcribing the operon, and (2) the low levels of cAMP prevent CAP from binding to the promoter and enhancing transcription .

When glucose is absent but lactose is present in the medium, E. coli switches to using lactose as its alternative carbon and energy source. In this case, the lac operon is induced by two mechanisms: (1) lactose (or more precisely, its isomer allolactose) binds to the lac repressor and inactivates it, allowing RNA polymerase to transcribe the operon, and (2) the high levels of cAMP bind to CAP and activate it, enabling it to bind to the promoter and facilitate transcription .

When both glucose and lactose are absent in the medium, E. coli does not need to express the lac operon at all. In this case, the lac operon is repressed by one mechanism: (1) the lac repressor binds to the operator and blocks RNA polymerase from transcribing the operon .

Catabolite repression is a type of positive control of transcription, since a regulatory protein (CAP) affects an increase (upregulation) in the rate of transcription of an operon. Catabolite repression allows E. coli to adapt to different environmental conditions and use the most efficient carbon and energy source available.

Attenuation is a mechanism of gene regulation that occurs in some prokaryotic operons, such as the trp operon and other amino acid biosynthetic operons. Attenuation takes advantage of the fact that in prokaryotes, transcription and translation occur simultaneously in the cytoplasm . This means that the ribosomes can start translating the mRNA while RNA polymerase is still transcribing the DNA sequence, and that the process of translation can directly affect transcription of the operon.

The working mechanism of attenuation involves a leader sequence, which is a short region of DNA upstream of the structural genes of the operon. The leader sequence contains a promoter, an operator, and a terminator. It also contains four regions (1-4) that can form complementary base pairs with each other and create different secondary structures in the mRNA.

When the operon is transcribed, region 1 is translated into a short peptide that contains two adjacent tryptophan codons. If tryptophan levels are high in the cell, the ribosomes can quickly translate region 1 and reach region 2. This allows region 3 to pair with region 4 and form a hairpin loop that acts as a termination signal for RNA polymerase. As a result, transcription of the operon is terminated before reaching the structural genes.

However, if tryptophan levels are low in the cell, the ribosomes stall at region 1 due to the lack of tryptophanyl-tRNA. This prevents region 2 from pairing with region 3, and instead allows region 2 to pair with region 1. This prevents region 3 from pairing with region 4, and instead allows region 3 to pair with region 2. This creates a different secondary structure in the mRNA that does not act as a termination signal for RNA polymerase. As a result, transcription of the operon continues and reaches the structural genes.

Therefore, attenuation regulates protein synthesis in prokaryotes by sensing the availability of tryptophan (or other amino acids) and controlling whether RNA polymerase can transcribe the entire operon or not. Attenuation is an example of how translation can influence transcription in prokaryotes.

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