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Learning Objectives Connection for AP® Courses The trp Operon: A Repressor Operon Catabolite Activator Protein (CAP): An Activator Regulator The lac Operon: An Inducer Operon Disclaimer

Learning Objectives

In this section, you will explore the following question:

What are operons and what are the roles of activators, inducers, and repressors in regulating operons and gene expression?

Connection for AP® Courses

Connection for AP^® Courses

The regulation of gene expression in prokaryotic cells occurs at the transcriptional level. Simply stated, if a cell does not transcribe the DNA’s message into mRNA, translation (protein synthesis), does not occur. Bacterial genes are often organized into common pathways or processes called operons for more coordinated regulation of expression. For example, in E. coli, genes responsible for lactose metabolism are located together on the bacterial chromosome. (The operon model includes several components, so when studying how the operon works, it is helpful to refer to a diagram of the model. See Figure 16.3 and Figure 16.4.) The operon includes a regulatory gene that codes for a repressor protein that binds to the operator, which prevents RNA polymerase from transcribing the gene(s) of interest. An example of this is seen in the structural genes for lactose metabolism. However, if the repressor is inactivated, RNA polymerase binds to the promoter, and transcription of the structural genes occurs.

There are three ways to control the transcription of an operon: inducible control, repressible control, and activator control. The lac operon is an example of inducible control because the presence of lactose turns on transcription of the genes for its own metabolism. The trp operon is an example of repressible control because it uses proteins bound to the operator sequence to physically prevent the binding of RNA polymerase. If tryptophan is not needed by the cell, the genes necessary to produce it are turned off. Activator control (typified by the action of Catabolite Activator Protein) increases the binding ability of RNA polymerase to the promoter. Certain genes are continually expressed via this regulatory mechanism.

Information presented and the examples highlighted in the section support concepts outlined in Big Idea 3 of the AP^® Biology Curriculum Framework. The Learning Objectives listed in the Curriculum Framework provide a transparent foundation for the AP^® Biology course, an inquiry-based laboratory experience, instructional activities, and AP^® exam questions. A Learning Objective merges required content with one or more of the seven Science Practices.

Big Idea 3	Living systems store, retrieve, transmit, and respond to information essential to life processes.
Enduring Understanding 3.B	Expression of genetic information involves cellular and molecular mechanisms.
Essential Knowledge	3.B.1 Gene regulation results in differential gene expression, leading to cell specialization
Science Practice	1.4 The student can use representations and models to analyze situations or solve problems qualitatively and quantitatively
Learning Objective	3.21 The student can use representations to describe how gene regulation influences cell products and function.
Essential Knowledge	3.B.2 A variety of intercellular and intracellular signal transmissions mediate gene expression.
Science Practice	1.4 The student can use representations and models to analyze situations or solve problems qualitatively and quantitatively.
Learning Objective	3.23 The student can use representations to describe mechanisms of the regulation of gene expression.

The DNA of prokaryotes is organized into a circular chromosome supercoiled in the nucleoid region of the cell cytoplasm. Proteins that are needed for a specific function, or that are involved in the same biochemical pathway, are encoded together in blocks called operons. For example, all of the genes needed to use lactose as an energy source are coded next to each other in the lactose (or lac) operon.

In prokaryotic cells, there are three types of regulatory molecules that can affect the expression of operons: repressors, activators, and inducers. Repressors are proteins that suppress transcription of a gene in response to an external stimulus, whereas activators are proteins that increase the transcription of a gene in response to an external stimulus. Finally, inducers are small molecules that either activate or repress transcription depending on the needs of the cell and the availability of substrate.

The trp Operon: A Repressor Operon

Bacteria such as E. coli need amino acids to survive. Tryptophan is one such amino acid that E. coli can ingest from the environment. E. coli can also synthesize tryptophan using enzymes that are encoded by five genes. These five genes are next to each other in what is called the tryptophan (trp) operon (Figure 16.3). If tryptophan is present in the environment, then E. coli does not need to synthesize it and the switch controlling the activation of the genes in the trp operon is switched off. However, when tryptophan availability is low, the switch controlling the operon is turned on, transcription is initiated, the genes are expressed, and tryptophan is synthesized.

The trp operon has a promoter, an operator, and five genes named trpE, trpD, trpC, trpB, and trpA that are located in sequential order on the DNA. RNA polymerase binds to the promoter. When tryptophan is present, the trp repressor binds the operator and prevents the RNA polymerase from moving past the operator; therefore, RNA synthesis is blocked. In the absence of tryptophan, the repressor dissociates from the operator. RNA polymerase can now slide past the operator, and transcription begins.

Figure 16.3 The five genes that are needed to synthesize tryptophan in E. coli are located next to each other in the trp operon. When tryptophan is plentiful, two tryptophan molecules bind the repressor protein at the operator sequence. This physically blocks the RNA polymerase from transcribing the tryptophan genes. When tryptophan is absent, the repressor protein does not bind to the operator and the genes are transcribed.

A DNA sequence that codes for proteins is referred to as the coding region. The five coding regions for the tryptophan biosynthesis enzymes are arranged sequentially on the chromosome in the operon. Just before the coding region is the transcriptional start site. This is the region of DNA to which RNA polymerase binds to initiate transcription. The promoter sequence is upstream of the transcriptional start site; each operon has a sequence within or near the promoter to which proteins (activators or repressors) can bind and regulate transcription.

A DNA sequence called the operator sequence is encoded between the promoter region and the first trp coding gene. This operator contains the DNA code to which the repressor protein can bind. When tryptophan is present in the cell, two tryptophan molecules bind to the trp repressor, which changes shape to bind to the trp operator. Binding of the tryptophan–repressor complex at the operator physically prevents the RNA polymerase from binding, and transcribing the downstream genes.

When tryptophan is not present in the cell, the repressor by itself does not bind to the operator; therefore, the operon is active and tryptophan is synthesized. Because the repressor protein actively binds to the operator to keep the genes turned off, the trp operon is negatively regulated and the proteins that bind to the operator to silence trp expression are negative regulators.

Link to Learning

Watch this video to learn more about the trp operon.

What would happen if bacteria did not have trp R?

The cell would not be able to break down tryptophan.
The cell will gradually produce more tryptophan over time.
The cell would not be able to make tryptophan.
The cell would make tryptophan when it was not needed.

Catabolite Activator Protein (CAP): An Activator Regulator

Just as the trp operon is negatively regulated by tryptophan molecules, there are proteins that bind to the operator sequences that act as a positive regulator to turn genes on and activate them. For example, when glucose is scarce, E. coli bacteria can turn to other sugar sources for fuel. To do this, new genes to process these alternate genes must be transcribed. When glucose levels drop, cyclic AMP (cAMP) begins to accumulate in the cell. The cAMP molecule is a signaling molecule that is involved in glucose and energy metabolism in E. coli. When glucose levels decline in the cell, accumulating cAMP binds to the positive regulator catabolite activator protein (CAP), a protein that binds to the promoters of operons that control the processing of alternative sugars. When cAMP binds to CAP, the complex binds to the promoter region of the genes that are needed to use the alternate sugar sources (Figure 16.4). In these operons, a CAP binding site is located upstream of the RNA polymerase binding site in the promoter. This increases the binding ability of RNA polymerase to the promoter region and the transcription of the genes.

The lac operon consists of a promoter, an operator, and three genes named lacZ, lacY, and lacA that are located in sequential order on the DNA. In the absence of cAMP, the CAP protein does not bind the DNA. RNA polymerase binds the promoter, and transcription occurs at a slow rate. In the presence of cAMP, a CAP–cAMP complex binds to the promoter and increases RNA polymerase activity. As a result, the rate of RNA synthesis is increased.

Figure 16.4 When glucose levels fall, E. coli may use other sugars for fuel but must transcribe new genes to do so. As glucose supplies become limited, cAMP levels increase. This cAMP binds to the CAP protein, a positive regulator that binds to an operator region upstream of the genes required to use other sugar sources.

The lac Operon: An Inducer Operon

The third type of gene regulation in prokaryotic cells occurs through inducible operons, which have proteins that bind to activate or repress transcription depending on the local environment and the needs of the cell. The lac operon is a typical inducible operon. As mentioned previously, E. coli is able to use other sugars as energy sources when glucose concentrations are low. To do so, the cAMP-CAP protein complex serves as a positive regulator to induce transcription. One such sugar source is lactose. The lac operon encodes the genes necessary to acquire and process the lactose from the local environment. CAP binds to the operator sequence upstream of the promoter that initiates transcription of the lac operon. However, for the lac operon to be activated, two conditions must be met. First, the level of glucose must be very low or non-existent. Second, lactose must be present. Only when glucose is absent and lactose is present will the lac operon be transcribed (Figure 16.5). This makes sense for the cell, because it would be energetically wasteful to create the proteins to process lactose if glucose was plentiful or lactose was not available.

Visual Connection

The lac operon consists of a promoter, an operator, and three genes named lacZ, lacY, and lacA. RNA polymerase binds to the promoter. In the absence of lactose, the lac repressor binds to the operator and prevents RNA polymerase from transcribing the operon. In the presence of lactose, the repressor is released from the operator, and transcription proceeds at a slow rate. Binding of the cAMP–CAP complex to the promoter stimulates RNA polymerase activity and increases RNA synthesis. However, even in the

Figure 16.5 Transcription of the lac operon is carefully regulated so that its expression only occurs when glucose is limited and lactose is present to serve as an alternative fuel source.

In E. coli, the trp operon is ON by default, while the lac operon is OFF. Why do you think that this is the case?

The trp operon is inducible and is positively regulated. Therefore it is ON by default whereas the lac operon is repressible and is OFF by default.
The trp operon synthesizes tryptophan which is essential for the cell and therefore remains ON, whereas the lac operon synthesizes enzymes for the breakdown of a sugar that is not always available and remains OFF by default.
The trp operon is constitutive and remains ON by default, whereas the lac operon is repressible and therefore is OFF by default.
The lac operon undergoes transcriptional attenuation and therefore is OFF by default, whereas the trp operon is not regulated by any such mechanism and is ON by default.

If glucose is absent, then CAP can bind to the operator sequence to activate transcription. If lactose is absent, then the repressor binds to the operator to prevent transcription. If either of these requirements is met, then transcription remains off. Only when both conditions are satisfied is the lac operon transcribed (Table 16.2).

Signals that Induce or Repress Transcription of the lac Operon
Glucose	CAP Binds	Lactose	Repressor Binds	Transcription
+	-	-	+	No
+	-	+	-	Some
-	+	-	+	No
-	+	+	-	Yes

Table 16.2

Link to Learning

Watch an animated tutorial about the workings of lac operon here.

The E. coli bacteria can have several mutations that affect the lac operon system. One mutation inhibits the ability of RNA polymerase to bind to the lac operon. How would this affect the cell?

The cell would make more lactose.
There would be no lactose outside of the cell.
The cell would not be able to process tryptophan.
The cell would not be able to process lactose.

Science Practice Connection for AP® Courses

Activity

Modeling the Operon. Use construction paper or more elaborate materials, such as Styrofoam noodles, electrical tape, and Velcro tabs, to create a model of the lac and trp operons that include a regulator, promoter, operator, and structural genes. Then use the model to show how the presence of substrate, for example, allolactose or tryptophan, can change the activity of the operons. As an extension of the activity, use the model to make predictions about the effects of mutations in any of the regions on gene expression.

Think About It

In E. coli, the trp operon is on by default, while the lac operon is off by default. Why do you think this is the case?

Disclaimer

This section may include links to websites that contain links to articles on unrelated topics. See the preface for more information.

16.2 Prokaryotic Gene Regulation

Learning Objectives