Chapter 14

Operator site

(also known simply as the operator) is a sequence of bases that provides a binding site for a repressor protein called the lac repressor.

FIGURE 14.5-The cycle of lac operon induction and repression.

**The lac repressor is bound to the lac operon when it is not exposed to lactose—when allolactose is not bound to the repressor.

FIGURE 14.3-Organization of the lac operon and other genes involved with lactose metabolism in E. coli.

**The lacZ, lacY, and lacA genes are under the control of the lac promoter.

How many operator sites for the lac repressor does the *lac operon* have?

3 operator sites

CAP site

A DNA sequence recognized by an activator protein called the catabolite activator protein (CAP).

Trans acting factor

A regulatory protein, such as the lac repressor, is called a trans-acting factor.

Promoter

For transcription to take place, an operon is flanked by a promoter that signals the beginning of transcription

Allolactose

allolactose acts as a small effector molecule for regulating the lac operon.

Negative control

repressor protein

Translation regulation

translational regulation typically require several minutes or even hours to take effect because this mechanism involve the synthesis and turnover of mRNA and polypeptides. **The 5´ region of an mRNA may exist in one conformation in which the Shine-Dalgarno sequence cannot be recognized by the ribosome, whereas the other conformation has an accessible Shine-Dalgarno sequence that allows the mRNA to be translated.**

Constitutive genes

unregulated genes have essentially constant levels of expression in all conditions over time. Unregulated genes are also called constitutive genes. Frequently, constitutive genes encode proteins that are continuously needed for the survival of the bacterium.

lac promoter

31 bp The promoter (lacP) is responsible for the transcription of the lacZ, lacY, and lacA genes as a single unit, which ends at the lac terminator.

Merozygote (Partial diploid)

A strain of bacteria containing Fʹ factor genes is called a merozygote, or partial diploid.

Terminator

For transcription to take place, an operon is flanked by a terminator that specifies the end of transcription.

Why do operons occur in bacteria?

One biological advantage of an operon organization is that it allows a bacterium to coordinately regulate a group of two or more genes that are involved with a common functional goal; the expression of the genes occurs as a single unit. For transcription to take place, an operon is flanked by a promoter that signals the beginning of transcription and a terminator that specifies the end of transcription. Two or more genes are found between these two sequences.

Polycistronic RNA

RNA that contains the sequences of two or more genes. Encoded by an operon

Allosteric regulation

The action of a small effector molecule, such as allolactose, is called allosteric regulation.

Transcription regulation

Transcriptional regulation by a repressor protein is termed negative control, and regulation by an activator protein is considered to be positive control. small effector molecules often play a critical role in transcriptional regulation. typically require several minutes or even hours to take effect because this mechanism involve the synthesis and turnover of mRNA and polypeptides. **The 5´ region of an mRNA may exist in one conformation that forms a ρ-independent terminator, which causes attenuation of transcription. The other conformation does not form a terminator and is completely transcribed.**

What are constitutive genes?

Unregulated genes are also called constitutive genes. Frequently, constitutive genes encode proteins that are continuously needed for the survival of the bacterium.

Induced

When four molecules of allolactose bind to the repressor, a conformational change occurs that prevents the lac repressor from binding to the operator site. Under these conditions, RNA polymerase is now free to transcribe the operon (Figure 14.4b). In genetic terms, we would say that the operon has been induced.

Cis acting element

a cis-acting element is a DNA segment that must be adjacent to the gene(s) that it regulates, and it is said to have a cis-effect on gene expression.

Repressible genes

a gene that is regulated by a corepressor or inhibitor, which are small effector molecules that cause transcription to decrease.

Repressor

a regulatory protein that binds to the DNA and inhibits transcription

Activator

a regulatory protein that increases the rate of transcription.

The *lac operon* is REGULATED by:

a repressor protein and an activator protein

Inducer

a small effector molecule that causes transcription to increase. may accomplish this in two ways: 1)It could bind to a repressor protein and prevent it from binding to the DNA 2)it could bind to an activator protein and cause it to bind to the DNA. In either case, the transcription rate is increased.

Which of the following conditions would cause transcription to be activated? a. A repressor plus an inducer b. A repressor plus a corepressor c. An activator plus an inhibitor d. None of the above

a. A repressor plus an inducer

Positive control

activator protein

What is an operon? a. A site in the DNA where a regulatory protein binds b. A group of genes under the control of a single promoter c. An mRNA that encodes several genes d. All of the above

b. A group of genes under the control of a single promoter

On its chromosome, an E. coli cell is lacI lacZ lacY lacA . It has an Fʹ factor that is lacI lacZ lacY lacA . What is the expected level of expression of the lac operon genes (lacZ lacY lacA ) in the absence of lactose? a. Both lac operons would be expressed. b. Neither lac operon would be expressed. c. Only the chromosomal lac operon would be expressed. d. Only the lac operon on the Fʹ factor would be expressed.

b. Neither lac operon would be expressed.

Cis effect

mediated by DNA sequences that are binding sites for regulatory proteins.

Post translation regulation

posttranslational regulation refers to the functional control of proteins that are already present in the cell rather than regulation of transcription or translation. Posttranslational control can either activate or inhibit the function of a protein. posttranslational control can be relatively fast, occurring in a matter of seconds, which is an important advantage.

The *lac operon* ENCODES:

proteins involved in Lactose Metabolism a diffusible repressor protein

The regulation of the *lac operon* allows a bacterium to:

respond to environmental change

Cyclic AMP (cAMP)

small effector molecule Produced from ATP via an enzyme known as adenylyl cyclase. When a bacterium is exposed to glucose, the transport of glucose into the cell stimulates a signaling pathway that causes the intracellular concentration of cAMP to decrease because the pathway inhibits adenylyl cyclase, the enzyme needed for cAMP synthesis. The effect of cAMP on the lac operon is mediated by an activator protein called the catabolite activator protein (CAP). CAP is composed of two subunits, each of which binds one molecule of cAMP.

Enzyme adaptation

the phenomenon in which a particular enzyme appears within a living cell after the cell has been exposed to the substrate for that enzyme

Gene regulation

the phenomenon in which the level of gene expression can vary under different conditions.

The phenomenon of enzyme adaptation is due to:

the synthesis of cellular proteins

F' factors

transfer of circular segments of DNA known as F factors. Sometimes an F factor also carries genes that were originally found within the bacterial chromosome. These types of F factors are called Fʹ factors (F prime factors). Monod, and Pardee identified Fʹ factors that carried the lacI gene and the lac operon. These Fʹ factors can be transferred from one cell to another by bacterial conjugation.

Explain how the lac operon is regulated by lac repressor and by catabolite activator protein

*The lac repressor is a protein that represses (inhibits) transcription of the lac operon. It does this by binding to the operator, which partially overlaps with the promoter. When bound, the lac repressor gets in RNA polymerase's way and keeps it from transcribing the operon. *When lactose is not available, the lac repressor binds tightly to the operator, preventing transcription by RNA polymerase. However, when lactose is present, the lac repressor loses its ability to bind DNA. It floats off the operator, clearing the way for RNA polymerase to transcribe the operon.

Explain how Jacob, Monod, and Pardee indicated that the lacI gene encodes a diffusible repressor protein

Because the normal lacI gene on the Fʹ factor was not physically located next to the chromosomal lac operon, this result is consistent with the idea that the lacI gene codes for a repressor protein that can diffuse throughout the cell and bind to any lac operon.

Inducible genes

Genes that are regulated in this manner (via Inducer) are called inducible genes.

Regulatory proteins

Genetic regulatory proteins that respond to small effector molecules typically have two binding sites. One site is where the protein binds to the DNA; the other is the binding site for the effector molecule. Regulatory proteins are given names describing how they affect transcription when they are bound to the DNA (repressor or activator).

Allosteric site

The functioning of allosteric proteins, such as the lac repressor, is controlled by effector molecules that bind to the proteins' allosteric sites.

Catabolite Repression

The lac operon is transcriptionally This form of transcriptional regulation is influenced by the presence of glucose, which is a catabolite—a substance that is broken down inside the cell. The presence of glucose ultimately leads to repression of the lac operon. When exposed to both glucose and lactose, E. coli cells first use glucose, and catabolite repression prevents the use of lactose.

Diauxic

The sequential use of two sugars by a bacterium, known as diauxic growth, is a common phenomenon among many bacterial species. Glucose, a more commonly encountered sugar, is metabolized preferentially, and then a second sugar is metabolized after glucose is depleted from the environment.

Some mutations have a cis-effect, whereas others have a transeffect. Explain the molecular differences between cis- and transmutations. Which type of mutation (cis or trans) can be complemented in a merozygote experiment?

A mutation that has a cis-effect is within a genetic regulatory sequence, such as an operator site, that affects the binding of a genetic regulatory protein. A cis-effect mutation affects only the adjacent genes that the genetic regulatory sequence controls. A mutation having a trans-effect is usually in a gene that encodes a genetic regulatory protein. A trans-effect mutation can be complemented in a merozygote experiment by the introduction of a normal gene that encodes the regulatory protein.

Transcriptional regulation often involves a regulatory protein that binds to a segment of DNA and a small effector molecule that binds to the regulatory protein. Do the following terms apply to a regulatory protein, a segment of DNA, or a small effector molecule? A. Repressor B. Inducer C. Operator site E. Activator G. Inhibitor

A- regulatory protein B- effector molecule C- DNA segment E- regulatory protein G- Effector molecule

In the lac operon, how would gene expression be affected if one of the following segments was missing? A. lac operon promoter B. Operator site C. lacA gene

A. No transcription would take place. The lac operon could not be expressed. B. No regulation would take place. The operon would be continuously turned on. C. The rest of the operon would function normally, but none of the transacetylase would be made.

What is the difference between a constitutive gene and a regulated gene?

Constituted gene: is unregulated, which means that its expression level is relatively constant. Expression of a regulated gene varies under different conditions In BACTERIA: the regulation of genes often occurs at the level of transcription by combinations of regulatory proteins and small effector molecules Gene expression can be regulated at the level of translation or the the function of a protein can be regulated after translation is completed

What is diauxic growth? Explain the roles of cAMP and the catabolite activator protein in this process.

Diauxic growth refers to the phenomenon in which a cell first uses up one type of sugar (such as glucose) before it begins to metabolize a second sugar (e.g., lactose). In this case, it is caused by gene regulation. When a bacterial cell is exposed to both sugars, the uptake of glucose causes the cAMP levels in the cell to fall. When this occurs, the catabolite activator protein (CAP) is removed from the lac operon, so it is not able to be activated by CAP.

Beta-galactosidase

Encoded by lacZ an enzyme that cleaves lactose into galactose and glucose. As a side reaction, β-galactosidase also converts a small percentage of lactose into allolactose, a structurally similar sugar

lacZ

LacZ encodes the enzyme β-galactosidase, an enzyme that cleaves lactose into galactose and glucose.

Permease

Lactose permease of E. coli is a protein composed of a single polypeptide that is 417 amino acids in length. By convention, the amino acids within a polypeptide are numbered from the aminoterminus to the carboxyl-terminus. **membrane protein required for the active transport of lactose into the cytoplasm of the bacterium.

Would a mutation that inactivated the lac repressor and prevented it from binding to the lac operator site result in the constitutive expression of the lac operon under all conditions? Explain. What is the disadvantage to the bacterium of having a constitutive lac operon?

A mutation that prevented the lac repressor from binding to the operator would make the lac operon constitutive only in the absence of glucose. However, this mutation would not be entirely constitutive because transcription would be inhibited in the presence of glucose. The disadvantage of constitutive expression of the lac operon is that the bacterial cell would waste a lot of energy transcribing the genes and translating the mRNA when lactose was not present.

Trans effect

A trans-effect is a form of genetic regulation that can occur even though two DNA segments are not physically adjacent. mediated by DNA sequences that are binding sites for regulatory proteins.

lacI gene (Repressor)

The lacI gene encodes the lac repressor, a protein that regulates the lac operon by binding to the operator site and repressing transcription. The lac repressor functions as a homotetramer, a protein composed of four identical subunits. Only a small amount of the lac repressor protein is needed to repress the lac operon.

I promoter

The lacI gene has its own promoter called the i promoter and is constitutively expressed at fairly low levels.

lacY

The lacY gene encodes lactose permease, a membrane protein required for the active transport of lactose into the cytoplasm of the bacterium.

What is enzyme adaptation? From a genetic point of view, how does it occur?

The term enzyme adaptation means that a particular enzyme is made only when a cell is exposed to the substrate for that enzyme. It occurs because the gene that encodes the enzyme that is involved in the metabolism of the substrate is expressed only when the cells have been exposed to the substrate

FIGURE 14.8- The roles of the lac repressor and catabolite activator protein (CAP) in the regulation of the lac operon.

**The repressor protein allows the cell to avoid turning on the operon in the absence of lactose. The activator protein allows the cell to choose between glucose and lactose.

Using three examples, describe how allosteric sites are important in the function of genetic regulatory proteins.

1. lac operon: the binding of allolactose causes a conformational change in the repressor protein and removes it from the operator site 2. lac operon: the binding of cAMP to CAP causes a conformational change that allows it to bind to the promoter region

If a gene is repressible and under positive control, describe what kind of effector molecule and regulatory protein are involved. Explain how the binding of the effector molecule affects the regulatory protein.

An inhibitor molecule and an activator protein are involved. The binding of the inhibitor molecule to the activator protein would prevent it from binding to the DNA and thereby inhibit its ability to activate transcription.

What is an advantage of gene regulation?

Gene regulation is more efficient. A cell doesn't waste energy making RNAs and proteins it doesn't need.

Describe the organization of the lac operon

The [promoter] is the binding site for RNA polymerase, the enzyme that performs transcription. The [operator] is a negative regulatory site bound by the lac repressor protein. The operator overlaps with the promoter, and when the lac repressor is bound, RNA polymerase cannot bind to the promoter and start transcription. The [CAP binding site] is a positive regulatory site that is bound by catabolite activator protein (CAP). When CAP is bound to this site, it promotes transcription by helping RNA polymerase bind to the promoter.

Examine figure 14.7: What is the function of B-ONPG? Explain the basis for the color differences in tubes 1-4

Add β-o-nitrophenylgalactoside (β-ONPG). This is a colorless compound. If β-galactosidase is present, it will cleave the compound to produce galactose and o-nitrophenol (O-NP). o-Nitrophenol has a yellow color. The deeper the yellow color, the more β-galactosidase was produced.

Mutations may have an effect on the expression of the lac operon and the trp operon. Would the following mutations have a cis- or trans-effect on the expression of the protein-encoding genes in the operon? A. A mutation in the operator site that prevents the lac repressor from binding to it B. A mutation in the lacI gene that prevents the lac repressor from binding to DNA

A: Cis-effect. It would affect only the genes that are in the adjacent operon B: Trans-effect. This is a mutation that affects a protein that can move throughout the cell

The binding of _______ to the lac repressor causes the lac repressor to _______ to the operator site, thereby _______ transcription. a. glucose, bind, inhibiting b. allolactose, bind, inhibiting c. glucose, not bind, increasing d. allolactose, not bind, increasing

d. allolactose, not bind, increasing

If an abnormal repressor protein could still bind allolactose but the binding of allolactose did not alter the conformation of the repressor protein, how would the expression of the lac operon be affected?

It would be impossible to turn the lac operon on even in the presence of lactose because the repressor protein would remain bound to the operator site

How does exposing an E. coli cell to glucose affect the regulation of the lac operon via CAP? a. cAMP binds to CAP and transcription is increased. b. cAMP binds to CAP and transcription is decreased. c. cAMP does not bind to CAP and transcription is increased. d. cAMP does not bind to CAP and transcription is decreased.

d. cAMP does not bind to CAP and transcription is decreased.

Operon

In bacteria, it is common for a few genes to be arranged together in an operon—a group of two or more genes under the transcriptional control of a single promoter. An operon encodes a polycistronic mRNA, an RNA that contains the sequences of two or more genes.

Lactose

1. The exposure of bacterial cells to lactose increased the levels of lactose-utilizing enzymes by 1000- to 10,000-fold. 2. Antibody and labeling techniques revealed that the increase in the activity of these enzymes was due to the increased synthesis of the proteins that form the enzymes. 3. The removal of lactose from the environment caused an abrupt termination in the synthesis of the enzymes. 4. The analysis of mutations in the lac operon revealed that each protein involved with lactose utilization is encoded by a separate gene. **enzyme adaptation is due to the synthesis of specific proteins in response to lactose in the environment.

In general, why is it important to regulate genes? Discuss examples of situations in which it would be advantageous for a bacterial cell to regulate genes.

In bacteria, gene regulation greatly enhances the efficiency of cell growth. It takes a lot of energy to transcribe and translate genes. Therefore, a cell is much more efficient and better at competing in its environment if it expresses genes only when the gene product is needed. For example, a bacterium will express only the genes that are necessary for lactose metabolism when a bacterium is exposed to lactose. When the environment is missing lactose, these genes are turned off. Similarly, when tryptophan levels are high within the cytoplasm, the genes required for tryptophan biosynthesis are repressed.

A repressor is a __________ that _________ transcription. a. small effector molecule, inhibits b. small effector molecule, enhances c. regulatory protein, inhibits d. regulatory protein, enhances

c. regulatory protein, inhibits

Effector molecules

small effector molecules often play a critical role in transcriptional regulation. small effector molecules do not bind directly to the DNA to alter transcription. an effector molecule exerts its effects by binding to a repressor or activator. The binding of the effector molecule causes a conformational change in the regulatory protein and thereby influences whether or not the protein can bind to the DNA.