Prokaryotic OperonsStandard 4.8: Gene regulation results in differential gene expression, leading to cell specialization.a. Describe the connection between the regulation of gene expression and observed differences between different kinds of organisms. d. Use representations to describe how gene regulation influences cell products and function.

Background1:In 1961 Francois Jacob and Jacques Monod discovered the process by which E. coli metabolizes lactose. This metabolism is controlled genetically in a gene expression system called an “operon”. Genes that code for proteins are called the structural gene (transcription unit) while controlling regions near the structural genes are called the operator and the promoter.

The operon complex contains the following parts:1. Promoter: The promoter is a region of DNA that initiates transcription upstream of the structural gene.

The promoter is recognized by the RNA polymerase and attaches there. 2. Operator: The operator controls the RNA polymerase access to the promoter, and is usually located

within the promoter or between the promoter and the transcribable gene. The operator essentially switches on or off the gene.

3. Structural Gene (transcription unit): The structural gene (or set of genes) codes for the needed polypeptide(s).

RepressorsRegulatory genes can be translated into repressor proteins. These function as an on/off switch for the operon. Repressor proteins work with controller molecules in the cell environment. Here is what they can do:

● A controller can activate a repressor, which then binds to the operator region of the gene, stopping, or repressing, gene activity. A controller that activates a repressor is sometimes called a co-repressor.

● A repressor may normally be sitting on the operator region of the gene. A controller can remove or deactivate the repressor, which activates, or induces, gene activity.

1 Rosemary Richardson, 2010 http://scidiv.bellevuecollege.edu/rkr/Biology211/lectures/pdfs/ProkaryGeneRegulation211S.pdf

Types of Operons● An inducible operon is one whose structural gene is normally off, blocked by its repressor. Its

controller substance attaches to the repressor molecule, removing it from blocking the gene (hence stopping its repression). The gene can then be "on", hence transcribed, until the repressor once again attaches to the gene's operator.

● A repressible operon is one whose gene is normally "on". When the controller substance attaches to the repressor molecule (that has not been attached to the operator), the repressor binds to the operator, blocking the structural gene and turning it off.

● In both inducible and repressible operons, the gene is off when the repressor is attached to its controller. The difference is where the repressor is located.

The Lactose Operon - InducibleIn the lactose operon, the substrate, allolactose (an isomer of lactose), attaches to a repressor protein that normally sits on the operator region of the gene. In the absence of lactose (the controller), the repressor sitting on the operator inhibits transcription by blocking RNA polymerase from attaching to the promoter. The gene is OFF.

When the substrate, allolactose (the controller), attaches to the repressor molecule that sits on the operator region of the gene, the repressor is removed from the gene. RNA polymerase can attach to the promoter and the genes that code for the three enzymes to digest lactose are transcribed.The Gene is ON.

The lactose inducible operon works in tandem with an activator, known as CAP (catabolite activator protein) that monitors the amount of glucose in the cell. Since glucose is the preferred fuel molecule in cell respiration, high levels of glucose in the cell block the lactose operon no matter how much lactose is present. There is no need to degrade lactose for fuel if there is plenty of glucose fuel available. This is also known as glucose repression, because the presence of glucose represses gene activity for other fuels.

Glucose in Environment

cAMP Levels

RNA PolymeraseBi

nding to Promoter?

Lactose in Environment

LAC Repressor

Transcription of LAC genes

Lactose Metabolized?

Present Low No Absent Bound to Operator Blocks

Polymerase

No No

Present Low Present but not efficient

Present Not Bound to Operator

Low Level No

However, when glucose levels are low in the cell, cAMP (cyclic adenosine monophosphate), an important secondary messenger in cell communications, accumulates. cAMP binds to the allosteric site of CAP forming a CAP-cAMP complex. CAP-cAMP binds to a site next to the lactose operon promoter and makes it easier for RNA polymerase to bind to the promoter region enhancing transcription of the lactase enzymes when lactose is present in the cell's environment to remove the lactose repressor molecule.

Glucose in Environment

cAMP Levels

RNA PolymeraseBi

nding to Promoter?

Lactose in Environment

LAC Repressor

Transcription of LAC genes

Lactose Metabolized?

Absent High Yes Present Not Bound to Operator

Yes Yes

Absent High No No Bound to Operator Blocks

Polymerase

No No

Lac Operon Animation ReviewPlease view the following short animations for the Lac operon. Please pause it as needed to understand the sequence of events occurring in the system.

● Lactose Operon

The Tryptophan Operon - RepressibleAll cells need the amino acid, tryptophan. Bacteria, such as E. coli, have the enzymes needed to synthesize tryptophan, and the genes that code for these enzymes are normally active. However, if tryptophan is in E. coli's environment, transcription is halted, and the enzymes needed to manufacture tryptophan in the bacterial cell are not synthesized.

A cellular product, in this case, tryptophan, can function to inhibit transcription, and is an example of the feedback inhibition common in cell homeostasis. High concentration of tryptophan stops the transcription of the set of enzymes that lead to tryptophan synthesis. Tryptophan does so by binding to an allosteric (non-active) site on the tryptophan repressor. This alters the shape of the repressor protein so that the operator blocks the attachment of RNA polymerase to the promoter.

The tryptophan operon is an example of gene regulation of repressible enzymes, because the presence of the product of the metabolic pathway represses (or stops) the synthesis of the enzyme(s) needed to synthesize it. Technically, the controller, tryptophan, is called a co-repressor because it works with the repressor protein to block transcription, and the tryptophan operon is a repressible operon. This is a negative control mechanism because the repressor blocks transcription.

Tryptophan in Environment

RNA Polymerase Binding to Promoter?

TRP Repressor Corepressor Transcription of TRP genes

Tryptophan Synthesized?

Absent Yes Not Bound to Operator

None Yes Yes

Present No Bound to Operator Blocks Polymerase

Activates Repressor

No No

Trp Operon Animation ReviewPlease view the following short animations for the Trp operon. Please pause it as needed to understand the sequence of events occurring in the system. Tryptophan Operon

Assessment of the Prokaryotic Operon 1. Complete the comparison of the lac Operon and trp Operons as a means of gene regulation.

lac operon trp operon

Regulates production of:

Number of genes and how they are controlled

What binds to the operator & when does this occur

High levels of what substance affects the operon how?

2. Why have genes under regulation? 3. What is the function of the promoter? 4. What is the function of the operator?

5. What happens if lactose levels are low? Put the following list in order (1-5).

RNA polymerase is blocked from transcribing the genes for the lactose metabolizing enzymes

When RNA polymerase binds to the promoter, it cannot get past the LacI repressor protein

The enzymes B-galactosidae, B-galacosidae permease, and transacetylase are not required by the cell due to low levels of lactose

Lactose does not bind to the repressor protein, LacI

LacI, a repressor protein, is bound to the operator, which follows the promoter

6. What happens if tryptophan levels are high? Put the following list in order (1-4).

The trp repressor-tryptophan complex can now bind to the operator of the trp operon

Tryptophan does not need to be produced by the trp operon

Tryptophan will bind to the repressor protein, changing its conformation

RNA Polymerase is blocked from transcribing the genes needed to synthesize tryptophan

7. What happens if lactose is present and glucose is scarce? Put the following list in order (1-7). Start with the repressor part first.

The three enzymes involved in the metabolism of lactose are transcribed and expressed

cAMP binds to CAP regulatory protein, causing it to bind to the promoter of the lac operon

The enzymes needed for lactose metabolism must be transcribed when lactose is present

cAMP levels increase because glucose is scarce (ATP is not being produced through cell respiration)

Lactose binds to the LacI repressor, changing LacI’s shape and making it fall off the operator

CAP binding causes RNA Polymerase to bind to the promoter (higher affinity) and transcribe the gene at a higher level than before

Now that LacI has been removed for the operator, RNA polymerase can proceed with transcription

Optional Modeling the Lac OperonYou can use a computer models of the Lac operon to further your understanding. The goal of this modeling activity is to learn how prokaryotes control gene expression.

PhET Lac Operon SimulationThe University of Colorado Boulder phET simulation allows you to manipulate a computer model of the Lac operon. To do this, you must download the simulator HERE.

Please follow the directions below2.

Step 1: Drag the lac promoter to the stretch of DNA. Do NOT drag the lacZ gene to the DNA. ● What happens? Why is this?

Step 2: Now try dragging the lacZ gene to the DNA and note what happens. Step 3: Inject some lactose (about 25 molecules should do it) into the simulation.

● Note what happens. Specifically, what is lactose being converted into? Step 4: Note that the lac enzyme continues to be produced even in the absence of lactose.

● Why is this a problem? ● Try dragging the lac operator gene onto the stretch of DNA. What is the result?

Step 5: Now try adding the lacI promoter and gene to the stretch of DNA. ● What happens?

Step 6: Again, add some lactose (and again, 25 molecules should work well) into the simulation. ● What is the INITIAL result of adding lactose when both genes are activated?

Step 7: Do not add any more lactose and watch what transpires. ● Note what happens and why this occurs. ● How could you re-activate the lacZ gene?

Step 8: Now try the lactose transport tab and insert all of the promoters and genes. Add some lactose and watch to see what transpires.

● What is the role of the lacY gene? ● How does this help the system?

2 D. LaFleur, 2015 http://phet-downloads.colorado.edu/files/activities/3568/Lac%20Operon%20AP%20Biology%20PhET%20Simulation.pdf