Chapter 18Chapter 18Regulation of Gene ExpressionRegulation of Gene Expression

Regulation of a Regulation of a Metabolic PathwayMetabolic Pathway

Bacterial often regulate Bacterial often regulate their metabolism based their metabolism based on what is present in on what is present in their environment.their environment.

Regulation can be Regulation can be controlled in two ways:controlled in two ways:

One, cell can adjust One, cell can adjust the activity of the activity of enzymes already enzymes already present through a present through a process called process called feedback inhibition.feedback inhibition.

Regulation of a Regulation of a Metabolic PathwayMetabolic Pathway

Bacterial often regulate Bacterial often regulate their metabolism based their metabolism based on what is present in on what is present in their environment.their environment.

Regulation can be Regulation can be controlled in two ways:controlled in two ways:

The second way The second way bacteria control their bacteria control their metabolism is by metabolism is by regulating transcription regulating transcription of the gene producing of the gene producing the enzyme. the enzyme.

The OperonThe OperonAn operon is a unit of genetic function common An operon is a unit of genetic function common to bacteria and phages that consist of to bacteria and phages that consist of coordinately controlled clusters of genes with coordinately controlled clusters of genes with similar functions. similar functions.

An operon consists of an An operon consists of an operatoroperator, a , a promoterpromoter, , and and the genes that code for the enzymesthe genes that code for the enzymes they they control in a particular pathway.control in a particular pathway.

The Basics of the The Basics of the OperonOperon

The The operatoroperator is the segment is the segment of the DNA that acts as a of the DNA that acts as a switch.switch.

A A repressorrepressor is a protein that is a protein that can bind to the operon and can bind to the operon and switch it off.switch it off.

The repressor is usually a The repressor is usually a product of a regulatory gene product of a regulatory gene often acting allosterically.often acting allosterically.

CorepressorsCorepressors are small are small molecules that cooperate with molecules that cooperate with repressor proteins to switch repressor proteins to switch operons off.operons off.

The Basics of the The Basics of the OperonOperon

InducersInducers are small are small molecules that molecules that inactivate repressors.inactivate repressors.

ActivatorsActivators are proteins are proteins that bind to DNA and that bind to DNA and stimulate transcription stimulate transcription of specific genes.of specific genes.

The Basics of the The Basics of the OperonOperon

Operons move back and forth between 2 states:Operons move back and forth between 2 states:

One with a repressor bound, and one without.One with a repressor bound, and one without.

Also, most regulatory proteins are allosterically Also, most regulatory proteins are allosterically controlled.controlled.

That is, there is an active state and an inactive That is, there is an active state and an inactive state.state.

Controlled by the amount of gene product Controlled by the amount of gene product available.available.

Inducible and Inducible and Repressible OperonsRepressible Operons

There are two main types of operons: There are two main types of operons: inducible and repressible. inducible and repressible.

Inducible operons are always off, but can be Inducible operons are always off, but can be “induced” to turn on when the products they “induced” to turn on when the products they make are needed.make are needed.

• The lac operon.The lac operon.

Repressible operons are always on, but can be Repressible operons are always on, but can be “repressed” when there is an abundance of “repressed” when there is an abundance of product they make.product they make.

• The trp operon.The trp operon.

Tryptophan: A Basic Tryptophan: A Basic ExampleExample

When the cell lacks tryptophan in its medium, a When the cell lacks tryptophan in its medium, a series of steps can be activated so that the cell series of steps can be activated so that the cell can synthesize it.can synthesize it.

When tryptophan is synthesized from a precursor When tryptophan is synthesized from a precursor molecule, a series of steps are activated and each molecule, a series of steps are activated and each is catalyzed by a specific enzyme.is catalyzed by a specific enzyme.

The 5 genes that code for the enzymes are The 5 genes that code for the enzymes are clustered together in a single stretch on the clustered together in a single stretch on the bacterial chromosome.bacterial chromosome.

A single promoter serves all 5 genes.A single promoter serves all 5 genes.

This constitutes a transcriptional unit, and when the This constitutes a transcriptional unit, and when the mRNA is synthesized, it codes for all five enzymes in mRNA is synthesized, it codes for all five enzymes in the pathway.the pathway.

The mRNA is punctuated with start and stop codons The mRNA is punctuated with start and stop codons that signal where the coding sequences begin and that signal where the coding sequences begin and end.end.

Thus, the 5 separate polypeptides can be translated Thus, the 5 separate polypeptides can be translated as needed.as needed.

Tryptophan: A Basic Tryptophan: A Basic ExampleExample

The advantage to this is that there is one on-off switch The advantage to this is that there is one on-off switch for the series of genes related to tryptophan synthesis.for the series of genes related to tryptophan synthesis.

When the cell needs tryptophan, all enzymes are When the cell needs tryptophan, all enzymes are switched on.switched on.

The “switch” is the segment of DNA called the The “switch” is the segment of DNA called the operator.operator.

The operator is positioned within or between the The operator is positioned within or between the promoter and the enzyme-coding genes.promoter and the enzyme-coding genes.

The operator controls the access of RNA polymerase to The operator controls the access of RNA polymerase to the genes.the genes.

Tryptophan: A Basic Tryptophan: A Basic ExampleExample

The The trptrp Operon, a Operon, a Repressible OperonRepressible Operon

The operon is The operon is switched off and on switched off and on by a protein called a by a protein called a repressor.repressor.

The operon, in this The operon, in this case, is always on case, is always on and binding of the and binding of the repressor blocks the repressor blocks the attachment of RNA attachment of RNA polymerase to the polymerase to the promoter and thus promoter and thus transcription of the transcription of the genes.genes.

This blocking action is This blocking action is very specific and has very specific and has no effect on other no effect on other genes within the cell.genes within the cell.

The The trptrp Operon, a Operon, a Repressible OperonRepressible Operon

In our example using tryptophan, the In our example using tryptophan, the trptrp repressor is repressor is the product of a regulatory gene (the product of a regulatory gene (trptrpRR) which is ) which is located some distance from the located some distance from the trptrp operon it is operon it is controlling.controlling.

It has its own promoter.It has its own promoter.

Regulatory genes are always on and always Regulatory genes are always on and always expressed within a cell. However, they bind expressed within a cell. However, they bind reversibly.reversibly.

The The trptrp Operon, a Operon, a Repressible OperonRepressible OperonIf the active genes If the active genes

ultimately produce ultimately produce tryptophan, and it’s tryptophan, and it’s available, it will bind available, it will bind to and alter the to and alter the function of the function of the trptrp repressor protein.repressor protein.

That is, it binds to That is, it binds to the protein, the protein, activating it and activating it and enabling it to bind to enabling it to bind to the operon shutting the operon shutting it off.it off.

Tryptophan is a Tryptophan is a corepressor.corepressor.

The The laclac Operon, An Operon, An Inducible OperonInducible Operon

The The laclac operon is an example of an inducible operon is an example of an inducible operon.operon.

ββ-galactosidase is an enzyme that breaks down -galactosidase is an enzyme that breaks down lactose into glucose and galactose.lactose into glucose and galactose.

Most Most E. coliE. coli cells only have a few molecules of cells only have a few molecules of ββ-galactosidase available at any one time.-galactosidase available at any one time.

When you add lactose to the medium in When you add lactose to the medium in which they are growing, the amount of which they are growing, the amount of ββ--galactosidase increases sharply.galactosidase increases sharply.

The gene encoding for The gene encoding for ββ-galactosidase is part of the -galactosidase is part of the laclac operon.operon.

It includes 2 other genes that code for enzymes that It includes 2 other genes that code for enzymes that function in lactose metabolism.function in lactose metabolism.

The whole thing is considered one transcriptional unit The whole thing is considered one transcriptional unit and is under the control of 1 operator and 1 promoter.and is under the control of 1 operator and 1 promoter.

The The laclac Operon, An Operon, An Inducible OperonInducible Operon

The The laclacII gene is a gene is a regulatory gene located regulatory gene located outside of the operon. It outside of the operon. It encodes for an allosteric encodes for an allosteric repressor protein that can repressor protein that can switch off the switch off the laclac operon operon by binding to the by binding to the operator.operator.

The reason it is called an The reason it is called an inducible operon is inducible operon is because the because the laclac repressor repressor is active by itself, and is active by itself, and binds to the operator and binds to the operator and shuts the shuts the laclac operon off. operon off.

The The laclac Operon, An Operon, An Inducible OperonInducible Operon

To inactivate the repressor, an inducer is needed. An To inactivate the repressor, an inducer is needed. An inducer is a small molecule which binds to and inducer is a small molecule which binds to and inactivates a repressor. inactivates a repressor.

In the case of the In the case of the laclac operon, allolactose is the operon, allolactose is the inducer. inducer.

Allolactose is an isomer of lactose found in small Allolactose is an isomer of lactose found in small amounts when lactose enters a cell.amounts when lactose enters a cell.

The The laclac Operon, An Operon, An Inducible OperonInducible Operon

When lactose is present, allolactose binds to the When lactose is present, allolactose binds to the repressor altering its conformation, preventing the repressor altering its conformation, preventing the ability of the repressor from binding to the operator. ability of the repressor from binding to the operator. When the repressor is unable to bind to the operator, When the repressor is unable to bind to the operator, the genes in the the genes in the laclac operon can be transcribed into operon can be transcribed into mRNA which can be translated into lactose-mRNA which can be translated into lactose-metabolizing enzymes. metabolizing enzymes.

The The laclac Operon, An Operon, An Inducible OperonInducible Operon

SummarySummary

The regulation of the The regulation of the trptrp and and laclac operons operons involves negative control of genes--their involves negative control of genes--their operons are switched off by the active form of operons are switched off by the active form of the repressor gene.the repressor gene.

Gene regulation is said to be under positive Gene regulation is said to be under positive control only when a regulatory protein control only when a regulatory protein interacts directly with the genome to switch interacts directly with the genome to switch transcription on. transcription on.

Positive Control of the Positive Control of the laclac OperonOperon

Not only are the lactose utilizing enzymes Not only are the lactose utilizing enzymes working and on when lactose is present, but they working and on when lactose is present, but they are also working when glucose is in short supply.are also working when glucose is in short supply.

E. coliE. coli cells can sense low concentrations of cells can sense low concentrations of glucose and relay the information to the genome.glucose and relay the information to the genome.

It does so with the interaction of an allosteric It does so with the interaction of an allosteric regulatory protein and a small organic molecule.regulatory protein and a small organic molecule.

Positive Control of the Positive Control of the laclac Operon Operon

When glucose is When glucose is scarce, cAMP scarce, cAMP accumulates in the accumulates in the cell. When cell. When accumulation occurs, accumulation occurs, it interacts with the it interacts with the regulatory protein regulatory protein called catabolite called catabolite activator protein activator protein (CAP).(CAP).

CAP acts as an CAP acts as an activator of activator of transcription.transcription.

Positive Control of the Positive Control of the laclac Operon Operon

When cAMP binds to When cAMP binds to CAP, CAP assumes an CAP, CAP assumes an active shape, binds to active shape, binds to a specific site on the a specific site on the laclac promoter promoter stimulating gene stimulating gene expression.expression.

This is called positive This is called positive regulation.regulation.

Positive Control of the Positive Control of the laclac Operon Operon

When glucose levels When glucose levels within the cell within the cell increase, cAMP falls increase, cAMP falls and CAP detaches and CAP detaches from the operon.from the operon.

When CAP is inactive, When CAP is inactive, transcription of the transcription of the laclac operon proceeds at a operon proceeds at a low level.low level.

SummarySummary

The state of the The state of the laclac repressor determines repressor determines whether or not transcription of the whether or not transcription of the laclac operon’s operon’s genes occurs at all. The state of CAP controls genes occurs at all. The state of CAP controls the rate of transcription if the operon is the rate of transcription if the operon is repressor free.repressor free.

In both cases, when glucose is available, it will In both cases, when glucose is available, it will be used preferentially by the cell. Lactose be used preferentially by the cell. Lactose enzymes will only be made when lactose is enzymes will only be made when lactose is available and glucose is not.available and glucose is not.

SummarySummary

In our example involving tryptophan, the In our example involving tryptophan, the trptrp operon, is a repressible operon because its operon, is a repressible operon because its transcription is usually on but can be turned transcription is usually on but can be turned off.off.

In contrast, the example involving lactose, the In contrast, the example involving lactose, the laclac operon, is an inducible operon. operon, is an inducible operon. Transcription is normally off, but can be turned Transcription is normally off, but can be turned on.on.

SummarySummaryWhen looking at When looking at the figure at the the figure at the right, keep in mind right, keep in mind the following 2 the following 2 things:things:

Negative control, Negative control, binding has a binding has a negative effect, it negative effect, it shuts down shuts down transcription.transcription.

Positive control, Positive control, binding has a binding has a positive effect, it positive effect, it stimulates stimulates transcription.transcription.