Module 16: Operons

Note from Mr. W: 12/27/16: Welcome! If you’re here in 2016, you’re one the first people to visit this page. If you have any questions about the content or the software, and, above all, if you see any problems or errors, please shoot me an email!

1. Mr. W’s Operons Slideshow

This is the presentation I give to my students to introduce them to how operons work. For more detail, jump down to the explanation below.

E. coli

2. Introduction: Why Genes need Regulation

Lactose

Escherichia coli (E. coli, for short) is an intestinal bacterium that inhabits the human colon, or large intestine, as well as the colons of many other mammals. Its genome consists of over 4000 protein-coding genes(wikipedia). While some of these proteins are constantly in use, others are only useful under certain conditions. For example, E. coli possesses a group of enzymes that it uses to digest the disaccharide lactose (the sugar in milk). If E. coli is in a lactose-rich environment, expressing the genes for lactose digesting enzymes will enable it to convert lactose into monosaccharides that can be further broken down during the reactions of cellular respiration. But if there’s no lactose, then producing the enzymes for lactose digestion would be a waste of energy. So, it’s of adaptive value for E. coli to be able to control its production of these enzymes to match the availability of lactose in the environment. How does E. coli do this?

3. Operons: Description and Definition

Many of E. coli’s genes (as well as many genes in other bacteria, archaea, viruses, and, more rarely, eukaryotic organisms) are organized into systems called operons. Operons consist of the following components.

The DNA at “4” consists of structural genes. These are the genes that code for enzymes or other gene products. These structural genes are under the control of a regulatory gene (1). The regulatory gene produces a regulatory protein that determines whether or not the structural genes will be transcribed into mRNA, which in turn is translated into protein. Because the regulatory protein often has the effect of preventing transcription, the regulatory protein is often referred to as a repressor.

Transcription is carried out by RNA polymerase, which can only transcribe a gene by binding with a region of DNA called the promoter, shown above at “2.” Just downstream of the promoter is a region called the operator (3). The operator is a binding site for the regulatory protein/repressor that the regulatory gene codes for. Pulling this together, we can define an operon as follows (modified from wikipedia):

…a cluster of genes under the control of a single promoter.The genes are transcribed and regulated in such a way that these genes are either expressed together or not at all.

This will become clearer by looking at an operon in action. We’ll start with the Lac operon, the first operon whose function was understood. This understanding came about through the work of François Jacob, André Michel Lwoff and Jacques Monod, who received the Nobel Prize for their work in 1965.

3. The Lac Operon

The Lac operon is the cluster of structural genes described above: they code for a series of enzymes that work together to convert lactose into two monosaccharides, glucose and galactose. Here’s how the expression of these structural genes is controlled.

The DNA at “1” is the regulatory gene. This gene is transcribed (process “a”) into mRNA (2), which in turn is translated (process “b”) into the regulatory protein (3).

The regulatory protein (also known as a repressor) has two binding sites. In the absence of lactose, one of its binding sites is complementary to the operator region (6) of the Lac operon. When the regulatory protein binds to the operator, RNA polymerase (4) can’t bind with its promoter (5), and, as a result, the structural genes (7) can’t be transcribed.

Below, you can see how the situation changes when lactose is in the environment.

Lactose (8) will bind with the regulatory protein in a way that causes an allosteric shift so that the regulatory protein’s DNA binding site is no longer complementary to the Lac operon’s operator (6). As a result, DNA polymerase can bind with its promoter, and will be able to transcribe the structural genes (7a, 7b, and 7c) into mRNA (9), which can, in turn, be translated into the enzymatic proteins that digest lactose.

4. Lac Operon: Labeled Diagrams

Got it? Prove it by labeling the interactive diagrams below.

[qwiz]

[h]Lac Operon: Interactive Diagrams

[i] Biohaiku

Operon System

Controls expression of genes

Such efficiency!

[!]Question 1 [/!]

[q labels = “top”]

 

 

[l]operator

[fx] No. Please try again.

[f*] Correct!

[l]promoter

[fx] No, that’s not correct. Please try again.

[f*] Good!

[l]regulatory gene

[fx] No. Please try again.

[f*] Great!

[l]regulatory mRNA 

[fx] No. Please try again.

[f*] Correct!

[l]regulatory protein

[fx] No, that’s not correct. Please try again.

[f*] Good!

[l]RNA polymerase

[fx] No. Please try again.

[f*] Correct!

[l]structural genes

[fx] No. Please try again.

[f*] Excellent!

[!]Question 2 BEGIN PASTE HERE!! [/!]

[q labels = “top”]

 

[l]operator

[fx] No. Please try again.

[f*] Good!

[l]promoter

[fx] No, that’s not correct. Please try again.

[f*] Good!

[l]regulatory gene

[fx] No. Please try again.

[f*] Excellent!

[l]regulatory mRNA 

[fx] No, that’s not correct. Please try again.

[f*] Excellent!

[l]regulatory protein

[fx] No, that’s not correct. Please try again.

[f*] Excellent!

[l]RNA polymerase

[fx] No. Please try again.

[f*] Excellent!

[l]structural genes

[fx] No, that’s not correct. Please try again.

[f*] Great!

[l]lactose

[fx] No. Please try again.

[f*] Correct!

[l]lactose-digesting enzymes

[fx] No, that’s not correct. Please try again.

[f*] Great!

[l]mRNA for lactose-digesting enzymes

[fx] No. Please try again.

[f*] Excellent!

[x][restart]
[/qwiz]

5. The Tryp Operon

Tryptophan: one of the 20 amino acids

An organism’s metabolic activities can be divided into categories: breaking things down (often to release energy, as in cellular respiration) and building things up. The Lac operon is in the first category: it produces enzymes for the breakdown of the disaccharide lactose into the two monosaccharides; glucose and galactose .

The Tryp operon is in the second category: it’s a system for controlling the synthesis of tryptophan, one of the 20 amino acids that make up proteins.

Because the Tryp operon involves synthesis, the logic behind its control is the opposite of that involved in the Lac operon. For the tryp operon, the logic is as follows: if tryptophan is lacking in the environment, then the operon system should be turned on, so that the cell can synthesize the tryptophan it needs. On the other hand, if tryptophan is available, then the operon should be turned off: instead of wasting energy and matter making tryptophan, the cell should simply let tryptophan diffuse in, and use it for whatever protein it’s synthesizing.

We’ll start with what happens when tryptophan is in E. coli’s environment (and thus diffusing into the cell). And in fact, based on what you know about the Lac operon, you should be able to figure out why, in this situation, the structural genes are not transcribed.

Here’s what’s happening. The regulatory gene (1) produces regulatory mRNA (2), which gets translated into a regulatory protein (3).

This regulatory protein has two binding sites. If tryptophan is present in the environment, it will bind with the regulatory protein, which causes a change in the regulatory protein’s DNA binding site. In this case, the conformation change is such that the regulatory protein can bind with the operator (6). This blocks RNA polymerase (4) from binding with the promoter (5), preventing transcription of the structural genes (7).

In the tyrp operon, the regulatory protein is often referred to as a repressor. Because the regulatory protein only binds to the operator when tryptophan is present, tryptophan is often referred to as a co-repressor. 

If tryptophan is not present in the environment, the situation changes as shown below.

Without tryptophan, the regulatory protein (3) can’t bind with the operator (6). RNA polymerase (4) is free to bind with the operon’s promoter (5) and transcribe the structural genes (7A through 7E), producing the structural mRNA (8A through 8E) that will be translated into the structural proteins (9A through 9E) that constitute the pathway for synthesizing tryptophan.

Got it? Label the diagrams below.

6. Tryp operon labeled diagrams

[qwiz]

[h]Tryp operon: Labeled Diagrams

[i]

[!]Question 1[/!!!]

[q labels = “top”]

 

 

[l]enzymes for tryptophan synthesis

[fx] No, that’s not correct. Please try again.

[f*] Correct!

[l]mRNA for structural genes

[fx] No. Please try again.

[f*] Good!

[l]operator

[fx] No. Please try again.

[f*] Good!

[l]promoter

[fx] No. Please try again.

[f*] Great!

[l]regulatory gene

[fx] No. Please try again.

[f*] Good!

[l]regulatory protein

[fx] No. Please try again.

[f*] Good!

[l]mRNA for regulatory protein

[fx] No, that’s not correct. Please try again.

[f*] Good!

[l]RNA polymerase

[fx] No. Please try again.

[f*] Good!

[l]structural genes

[fx] No. Please try again.

[f*] Good!

[!]Question 2 [/!!!]

[q labels = “top”]

 

 

[l]operator

[fx] No, that’s not correct. Please try again.

[f*] Good!

[l]promoter

[fx] No, that’s not correct. Please try again.

[f*] Correct!

[l]regulatory gene

[fx] No. Please try again.

[f*] Correct!

[l]regulatory gene’s mRNA

[fx] No. Please try again.

[f*] Great!

[l]regulatory protein

[fx] No. Please try again.

[f*] Good!

[l]RNA polymerase

[fx] No, that’s not correct. Please try again.

[f*] Good!

[l]structural genes

[fx] No. Please try again.

[f*] Excellent!

[l]tryptophan

[fx] No. Please try again.

[f*] Good!

[/qwiz]

7. Operons Quiz

This quiz uses diagrams and multiple choice questions to test your understanding of operons. Good luck!

[qwiz use_dataset=”Operons” random = “true” dataset_intro=”false”]

[h]Operons

[i]Biohaiku

An operator

Blocked by repressor protein

Prevents transcription

 

 

[/qwiz]

8. ADVANCED TOPIC: CAP (Catabolite Activator Protein) and Positive Gene Regulation

For the purposes of the AP Biology exam, the material on operons shown above is probably sufficient. But there’s another level to understanding prokaryotic gene regulation: If you’re interested, go for it!

We’ve seen above how the Lac operon allows E. coli to turn on production of enzymes to digest lactose when lactose is in its environment, and to turn production of these same enzymes off when lactose is not in the environment. But there’s an even more subtle regulatory scheme. If an E. coli cell is simultaneously fed with both lactose and glucose, it will preferentially use glucose, and only turn to lactose when glucose is short supply. From an energetic perspective, this makes sense. Glucose is the simplest carbohydrate: the genes for metabolizing it are always turned “on.” Lactose, on the other hand,  requires special enzymes to process. Given the choice, it’s energetically favorable for the cell to break down glucose first. But what system allows E. coli to express this preference?

The preference for glucose is controlled through expression of another molecule: cyclic AMP (cAMP). We’ve met cAMP before, as a key second messenger in cell communication. In the context of operons, here’s how cAMP works.

Let’s consider a situation where both glucose (shown below at letter “J”) and lactose (shown below at “G”) are present in the cell’s environment.

Under these circumstances, another regulatory protein called Catabolite Activator Protein (CAP, shown at “C” above) has a shape that doesn’t allow it to bind with the Lac Promoter (“E”) at the CAP binding site (“D”). CAP enhances the ability of RNA polymerase to bind with the promoter. So even though lactose (“G”) is present, and even though the Lac regulatory protein (“H”)  won’t bind with the Lac operator (“I”), very little of the structural genes will be transcribed. Glucose, not lactose, is the fuel that will be metabolized.

But if the glucose level drops and the lactose level stays high, then everything changes.

The low level of glucose induces production of cAMP (“B”). cAMP binds with CAP (at “C”) changing its conformation in such a way that it 1) binds with the promoter region of the lac operon at the CAP binding site (“D”). This binding enhances RNA polymerase’s ability to bind with the promoter (“E”). And with the regulatory protein (“H”) allosterically altered so that it won’t bind with the operator (“I”) RNA polymerase can transcribe the structural genes (7) for lactose digesting enzymes (not shown above).

As the authors of Campbell Biology conclude

Thus the lac operon is under dual control: negative control by the lac repressor and positive control by CAP. The state of the lac repressor…determines whether or not transcription occurs…; the state of CAP controls the rate of transcription…(Campbell Biology, 9th edition, page 355).

Got it? Label the diagrams below.

9. cAMP/CAP labeled diagrams

[qwiz]

[h]Positive Gene Regulation in Prokaryotes: cAMP and CAP

[i]

[q labels = “top”]

 

[l]CAP binding site

[fx] No, that’s not correct. Please try again.

[f*] Excellent!

[l]Catabolite Activator Protein

[fx] No, that’s not correct. Please try again.

[f*] Excellent!

[l]glucose

[fx] No. Please try again.

[f*] Excellent!

[l]lactose

[fx] No. Please try again.

[f*] Correct!

[l]operator

[fx] No. Please try again.

[f*] Correct!

[l]Promoter

[fx] No, that’s not correct. Please try again.

[f*] Excellent!

[l]regulatory gene

[fx] No, that’s not correct. Please try again.

[f*] Good!

[l]regulatory protein

[fx] No, that’s not correct. Please try again.

[f*] Excellent!

[l]RNA polymerase

[fx] No, that’s not correct. Please try again.

[f*] Great!

[l]structural genes

[fx] No. Please try again.

[f*] Correct!

 

[!!!]QUESTION 2 [/!!!!]

[q labels = “top”]

 

[l]CAP binding site

[fx] No. Please try again.

[f*] Good!

[l]Catabolite Activator Protein (inactive)

[fx] No, that’s not correct. Please try again.

[f*] Good!

[l]Catabolite Activator Protein (active)

[fx] No, that’s not correct. Please try again.

[f*] Great!

[l]lactose

[fx] No, that’s not correct. Please try again.

[f*] Excellent!

[l]operator

[fx] No. Please try again.

[f*] Excellent!

[l]Promoter

[fx] No. Please try again.

[f*] Good!

[l]regulatory gene

[fx] No. Please try again.

[f*] Correct!

[l]regulatory protein

[fx] No, that’s not correct. Please try again.

[f*] Great!

[l]RNA polymerase

[fx] No, that’s not correct. Please try again.

[f*] Great!

[l]structural genes

[fx] No. Please try again.

[f*] Correct!

[l]cAMP

[fx] No. Please try again.

[f*] Great!

[/qwiz]

10. Links