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CAP site (catabolite activator protein CAP)

a DNA sequence that is recognized by CAP; an abbreviation for the catabolite activator protein, a genetic regulatory protein found in bacteria

catabolite

a substance that is broken down inside the cell

allosteric sites

the site on a protein where a small effector molecule binds to regulate the function of the protein

trpR trpL

two genes that play a role in the regulation of the trp operon

the lac operon is regulated by a repressor protein

1) no lactose in the environment: in the absence of the inducer allolcatose, the repressor protein is tightly bound to the operator site, thereby inhibiting the ability of RNA polymerase to transcribe the operon 2) lactose present: when allolactose is available, it binds to the repressor, this alters the conformation of the repressor protein, which prevents it from binding to the operator site, therefore RNA polymerase can transcript the operon when the four molecules of allolactose bind to the repressor, a conformational change occurs that prevents the lac repressor form binding to the operator site

F' factors

F factors that also carry genes that were originally within the bacterial chromosome These F′ factors can be transferred from one cell to another by bacterial conjugation. A strain of bacteria containing F′ factor genes is called a merozygote, or partial diploid.

how the induction process is shut off when lactose in the environment has been depleted

The intracellular concentration of allolactose remains high as long as lactose is available in the environment. However, when lactose is depleted from the environment, the concentration of allolactose also becomes lower due to the action of metabolic enzymes. Eventually, the concentration of allolactose drops below its affinity for the repressor. At this point, allolactose is unlikely to be bound to lac repressor. When allolactose is released, lac repressor returns to the conformation that binds to the operator site. In this way, the binding of the repressor shuts down the lac operon when lactose is depleted from the environment. After repression occurs, the mRNA and proteins encoded by the lacoperon are eventually degraded (see Figure 14.5).

14.3 comprehension questions 1. When tryptophan binds to trp repressor, this causes trp repressor to __________ to the trp operator and ________ transcription. a. bind, inhibit b. not bind, inhibit c. bind, activate d. not bind, activate

a

inducible genes

a gene that is regulated by an inducer, which is a small effector molecule that causes transcription to increase

attenuator sequence

a sequence found in certain operons (e.g., the trp operon) in bacteria that stops transcription soon after it has begun.

cis-acting element

a sequence of DNA, such as a regulatory element, that exerts a cis-effect 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. The lacoperator site is an example of a cis-acting element. A trans-effect is mediated by genes that encode regulatory proteins, whereas a cis-effect is mediated by DNA sequences that are binding sites for regulatory proteins

corepressor

a small effector molecule that binds to a repressor protein, thereby causing the repressor protein to bind to DNA and inhibit transcription

operon

an arrangement in DNA in which two or more genes are found within a regulatory unit that is under the transcriptional control of a single promoter

cis-effect

an effect on gene expression due to a genetic sequence within the same chromosome and often immediately adjacent to the gene of interest

trans-effect

an effect on gene expression that occurs even though two DNA segments are not physically adjacent to each other. Trans-effects are mediated through diffusible genetic regulatory proteins The action of lac repressor on the lac operon is a trans-effect

polycistronic mRNA

an mRNA transcribed from an operon that encodes two or more proteins

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

benefit of operons in bacteria?

LacI gene

not part of the lac operon has its own "i" promoter expressed at low levels lacI gene encodes lac repressor, a protein that regulates the lac operon by binding to the operator site and repressing transcription. This repressor functions as a homotetramer, a protein composed of four identical subunits. Only a small amount of lac repressor is needed to repress the lac operon

Francois Jacob and Jacques Monod

provided an initial understanding of gene regulation

trpE trpD trpC trpB trpA

the five genes trp operon contains encode enzymes involved in tryptophan biosynthesis

enzyme adaptation

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

allosteric regulation

the phenomenon in whichPage G-2 an effector molecule binds to a noncatalytic site on a protein and causes a conformational change that regulates its function

gene regulation

the phenomenon whereby the level of gene expression can vary under different conditions the majority of genes are regulated, so the proteins they encode can be produced at the proper times and in the proper amounts the cell avoids wasting valuable energy making proteins it does not need

trpL

this region mediates attenuation A segment of DNA, termed the attenuator sequence, is important in facilitating this termination. When attenuation occurs, the mRNA from the trp operon is made as a short piece that terminates shortly past the trpL gene (see Figure 14.11c). Because this short mRNA has been terminated before RNA polymerase has transcribed the trpE, trpD, trpC, trpB, and trpA genes, it will not encode the proteins required for tryptophan biosynthesis. In this way, attenuation inhibits the further production of tryptophan in the cell. the first gene in the trp operon is the trpL gene

negative control

transcriptional regulation by a repressor protein

14.3 comprehension questions 3. Operons involved with the biosynthesis of molecules such as amino acids are most likely to be regulated in which of the following ways? a. The product of the biosynthetic pathway represses transcription. b. The product of the biosynthetic pathway activates transcription. c. A precursor of the biosynthetic pathway represses transcription. d. A precursor of the biosynthetic pathway activates transcription.

a

constitutive genes (unregulated genes)

a gene that is not regulated and has essentially constant levels of expression over time these genes encode proteins that are continuously needed for the survival of the bacterium

repressible genes

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

trpR

a gene that plays a role in the regulation of the trp operon has its own promoter and is not part of the trp operon encodes the trp repressor protein

merozygote (partial diploid)

a partial diploid strain of bacteria containing F′ factor genes

lac repressor

a protein that binds to the operator site of the lac operon and inhibits transcription

trp repressor

a protein that binds to the operator site of the trp operon and inhibits transcription When tryptophan levels within the cell are very low, trp repressor cannot bind to the operator site. Under these conditions, RNA polymerase transcribes the trp operon, and the cell expresses the genes required for the synthesis of tryptophan (see Figure 14.11a). When the tryptophan levels within the cell become high, tryptophan acts as a corepressor that binds to trp repressor. This causes a conformational change in trp repressor that allows it to bind to the trp operator site (Figure 14.11b). This inhibits the ability of RNA polymerase to transcribe the operon. Therefore, when a high level of tryptophan is present within the cell—when the cell does not need to make more tryptophan—the trp operon is turned off.

effector molecule

the three types: inducer, corepressor, and inhibitor do not bind directly other DNA to alter transcription, exerts its effects by binding to a repressor or activator the binding this causes a conformational change in the regulatory protein and thereby influences whether or not the protein can bind to the DNA 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.

14.3 comprehension questions 2. During attenuation, when tryptophan levels are high, the ________ stem-loop forms and transcription _________ the trpL gene. a. 1-2, ends just past b. 3-4, ends just past c. 1-2, continues beyond d. 3-4, continues beyond

b

activator

a regulatory protein that binds to DNA and increases the rate of transcription

repressor

a regulatory protein that binds to DNA and inhibits transcription

trans-acting factor

a regulatory protein that binds to a regulatory element in DNA and exerts a trans-effect A regulatory protein, such as lac repressor

inhibitor

a small effector molecule that binds to an activator protein, causing the protein to be released from the DNA, thereby inhibiting transcription

LacI-

a type of mutant strain of bacteria resulted in the expression of the lac operon even in the absence of lactose a loss-of-function mutation in the lacI gene prevented lac repressor from binding to the lac operator site and inhibiting transcription Alternative hypotheses to explain how a lacI− mutation could cause the constitutive expression of the lac operon. (a) The correct explanation in which the lacI− mutation eliminates the function of lac repressor, which prevents it from repressing the lac operon. (b, incorrect according to book..?) The hypothesis of Jacob, Monod, and Pardee. In this case, the lacI− mutation results in the synthesis of an internal activator that turns on the lac operon.

diauxic growth

definition: the sequential use of two sugars by a bacterium When exposed to both glucose and lactose, E. coli cells first use glucose, and catabolite repression prevents the use of lactose. Why is this an advantage? The explanation is efficiency. The bacterium does not have to express all of the genes necessary for both glucose and lactose metabolism. If the glucose is used up, catabolite repression is alleviated, and the bacterium then expresses the lac operon

the level of gene expression is increased or decreased, meaning the rate of RNA synthesis can be increased or decreased

instead of saying genes can be turn on or off, it is more accurate to say...

14.4 comprehension questions 2. An example of a posttranslational covalent modification that may regulate protein function is a. phosphorylation. b. acetylation. c. methylation. d. all of the above.

d

the cycle of lac operon induction and repression

the genes of the lac operon provide the bacterium with the trait of being able to metabolize lactose in the environment. When lactose is present, the genes of the lac operon are induced, and the proteins needed for the efficient uptake and metabolism of lactose are synthesized. When lactose is absent, these genes are repressed so the bacterium does not waste its energy expressing them. Note: The proteins involved with lactose utilization are fairly stable, but they will eventually be degraded

14.4 comprehension questions 1. Translation can be regulated by a. translational repressors. b. antisense RNA. c. attenuation. d. both a and b.

d

catabolite repression

the phenomenon in which a catabolite (e.g., glucose) represses the expression of certain genes (e.g., the lac operon) 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. Why is this an advantage? The explanation is efficiency. The bacterium does not have to express all of the genes necessary for both glucose and lactose metabolism. If the glucose is used up, catabolite repression is alleviated, and the bacterium then expresses the lac operon. The sequential use of two sugars by a bacterium, known as diauxic growth

terminator

a sequence within a gene that signals the end of transcription

allolactose

an inducer binds to the lac repressor, causing the repressor to come off the operator site, RNA polymerase creates mRNA, and the lac operon proteins are synthesized

14.2 comprehension questions 1. 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

transcription (gene --> mRNA) translation (mRNA --> protein) post translation (protein --> functional protein) which is all of the steps in the pathway of gene expression

gene regulation can occur at any of these steps in the pathway of gene expression

positive control

transcriptional regulation by an activator protein

under what conditions is lac repressor bound to the lac operon?

when there is minimal lactose in the environment the repressor is bound to the operator, very small amount of transcription will occur, thus small amounts of B-galactosidase, lactose permeate, and galactoside transacetylase are made. But this is too small of an amount for the bacterium to use lactose

14.5 comprehension question 1. For a riboswitch that controls transcription, the binding of a small molecule such as TPP controls whether the RNA a. has an antiterminator or terminator stem-loop. b. has a Shine-Dalgarno antisequestor or the Shine-Dalgarno sequence within a stem-loop. c. is degraded from its 5′ end. d. has an antiterminator or terminator stem-loop and has a Shine-Dalgarno antisequestor or the Shine-Dalgarno sequence within a stem-loop.

a

14.2 comprehension questions 3. On its chromosome, an E. coli cell has a genotype of lacI− lacZ+ lacY+ lacA+. It has an F′ factor with a genotype of lacI+ lacZ+ lacY+ lacA+. What is the expected level of expression of the laco peron genes (lacZ+ lacY+ lacA+) in the absence of lactose? a. Both lac operons will be expressed. b. Neither lac operon will be expressed. c. Only the chromosomal lac operon will be expressed. d. Only the lac operon on the F′ factor will be expressed

b

operator site

a sequence of nucleotides in bacterial DNA that provides a binding site for a genetic regulatory protein

promoter

a sequence within a gene that initiates (i.e., promotes) transcription

14.1 comprehension question 2. Which of the following combinations will 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

lac operon has three operator sites for lac repressor

O1 located just downstream from promoter O2 located downstream from promoter O3 located just upstream from promoter O2 and O3 originally pseudo-operators, but when they are missing repression is dramatically reduced even when O1 is present when all three operator sites are present the lac operons function is dramatically reduced, compared to the level of expression of an induced lac operon the binding of lac repressor to two operator sites requires the DNA to form a loop. A loop in the DNA brings the operator sites closer together, thereby facilitating the binding of the repressor protein (figure 14.10a) -The formation of this loop dramatically inhibits the ability of RNA polymerase to slide past the O1 site and transcribe the operon

14.1 comprehension question 1. 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

14.2 comprehension question 4. 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

14.2 comprehension questions 2. The binding of _______ to lac repressor causes 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

inducer

refers to a gene that has been transcriptionally activated by an inducer can bind to a repressor protein and prevent it from binding to the DNA, or it can bind to an activator protein and cause it to bind to the DNA

LacI gene

two lacI genes in a merozygote may be different alleles the genes on the bacterial chromosome and the genes on the F′ factor are not physically adjacent to each other

cyclic-AMP (cAMP)

in bacteria, a small effector molecule that binds to CAP (catabolite activator protein). In eukaryotes, cAMP functions as a second messenger in a variety of intracellular signaling pathways 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 the activator protein called catabolite activator protein (CAP). CAP is composed of two subunits, each of which binds one molecule of cAMP. When only lactose is present, allolactose and cAMP levels are high (Figure 14.8a). Allolactose binds to lac repressor and prevents it from binding to the DNA. The cAMP binds to CAP, and then CAP binds to the CAP site. A domain in CAP interacts with RNA polymerase, which facilitates the binding of RNA polymerase to the promoter. Under these conditions, transcription proceeds at a high rate. In the absence of both lactose and glucose, cAMP levels are also high (Figure 14.8b). However, the binding of lac repressor inhibits transcription even though CAP is bound to the DNA. Therefore, the transcription rate is very low.

attenuation

trp operon is regulated by this mechanism called attenuation a mechanism of genetic regulation, seen in the trp operon, in which a short RNA is made but its synthesis is terminated before RNA polymerase can transcribe the rest of the operon mediated by the region that includes the trpL gene One key feature of attenuation is that two tryptophan (Trp) codons are found within the mRNA. As we will see later, these two codons provide a way for the bacterium to sense whether or not it has sufficient tryptophan to synthesize its proteins. A second key feature that underlies attenuation is that the mRNA made from the trpL gene has four regions that are complementary to each other, which causes the mRNA to form stem-loops. Different combinations of stem-loops are possible due to interactions Page 350among these four regions (see the color key in Figure 14.12). Region 2 is complementary to region 1 and also to region 3. Region 3 is complementary to region 2 as well as to region 4. Therefore, three stem-loops are possible: 1-2, 2-3, and 3-4. Even so, keep in mind that a particular segment of RNA can participate in the formation of only one stem-loop. For example, if region 2 forms a stem-loop with region 1, it cannot (at the same time) form a stem-loop with region 3. Alternatively, if region 2 forms a stem-loop with region 3, then region 3 cannot form a stem-loop with region 4. Though three stem-loops are possible, the 3-4 stem-loop is functionally unique. The 3-4 stem-loop together with the U-rich attenuator sequence results in intrinsic termination, also called ρ-independent termination, as described in Chapter 12. Therefore, the formation of the 3-4 stem-loop causes RNA polymerase to pause, and the U-rich sequence dissociates from the DNA. This terminates transcription at the U-rich attenuator. In comparison, if region 3 forms a stem-loop with region 2, transcription will not be terminated because a 3-4 stem-loop cannot form. Conditions that favor the formation of the 3-4 stem-loop rely on the translation of the trpL gene and on the amount of tryptophan in the cell. As shown in Figure 14.13, three scenarios are possible. On some occasions, translation is not coupled with transcription (Figure 14.13a). As the trpL gene is being transcribed, region 1 rapidly hydrogen bonds to region 2, and region 3 is left to hydrogen bond to region 4. Therefore, the terminator stem-loop forms, and transcription is terminated just past the trpL gene at the U-rich attenuator.