chapter 16

Fill in the blanks in this table with "yes" or "no" for each condition of lac operon regulation. The strain is wild type, with no partial diploidy. The first line is filled in for reference

.

cis

A promoter that affects only genes that are on the same piece of DNA is referred to as a ____-acting promoter. a. cisb. transc. enhancer d. positive e. negative

These are mutations that are, respectively, cis- and trans-acting on lac operon expression.

If there are mutations that inactivate lacP and lacI, which of the following is TRUE? (I = lac repressor gene; Z, Y, A = lac operon structural genes; P = lac promoter; O = lac operator) These are mutations that are, respectively, cis- and trans-acting on lac operon expression. These are mutations that are, respectively, trans- and cis-acting on lac operon expression. These will affect the expression of I only. These will affect the expression of only Z, Y, and A. These mutations will have no effect.

the tryptophan level inside the bacterial cell is high.

The formation of 1+2 and 3+4 secondary structures of 5′ UTR region mRNA from the trp operon is triggered when the tryptophan level inside the bacterial cell is extremely low. the tryptophan level inside the bacterial cell is high. the repressor protein fails to bind to the operator. there is a spontaneous mutation introduced into the 5′ UTR. the structural gene transcription within the trp operon gets initiated.

attenuation

Transcriptional control that acts by regulating the continuation of transcription is called riboswitching. antitermination. negative control. operator mutation. attenuation.

Inducer

What is the function of allolactose in regulation of the lac operon? Inducer Repressor Activator Promoter Regulatory protein

Structural gene

Which of the following generally get transcribed constitutively? Regulatory gene Structural gene Operator element Promoter element Operon

I, Z, Y, A

Which parts of the DNA region shown in the diagram encode proteins? (I = lac repressor gene; Z, Y, A = lac operon structural genes; P = lac promoter; O = lac operator) P I, P, O P, O, Z, Y, A I, Z, Y, A I, P, O, Z, Y, A

repressor

The________ is a type of regulator protein that binds to a region of DNA in the promoter of a gene called the operator and prevents transcription from taking place. a. inducerb. repressor c. activator d. inactivator e. terminator

allosteric

When binding of the inducer to the repressor causes a conformational change, which then prevents the repressor from binding to DNA, the repressor is called a(n) __________ protein. a. coactivator b. allosteric c. structural d. operating e. responsive

3+4

Which of the following secondary structures causes attenuation of structural genes of the trp operon? a. 1+2 b. 1+3 c. 2+4 d. 2+3 e. 3+4

lacI+ lacP- lacO+ lacZ+ lacY+ lacA+

A mutant E. coli strain, grown under conditions that normally induce the lac operon, does not produce ß-galactosidase. What is a possible genotype of the cells? a. lacI+ lacP+ lacO+ lacZ+ lacY- lacA+ b. lacI+ lacP+ lacOc lacZ+ lacY+ lacA+ c. lacl+ lacP+ lacO+ lacZ+ lacY+ lacA+ d. lacI+ lacP- lacO+ lacZ+ lacY+ lacA+ e. lacI_ lacP+ lacO—lacZ+ lacY+ lacA-

lacI+ lacP+ lacOc lacZ+ lacY+ lacA+

A mutant E. coli strain, grown under conditions that normally induce the lac operon, produces high amounts of ß-galactosidase. What is a possible genotype of the cells? a. lacI+ lacP+ lacO+ lacZ- lacY+ lacA+ b. lacI+ lacP+ lacOc lacZ+ lacY+ lacA+ c. lacI- lacP+ lacO+ lacZ- lacY+ lacA+ d. lacI+ lacP- lacO+ lacZ+ lacY+ lacA+ e. lacI- lacP- lacO+ lacZ- lacY+ lacA-

The extra piece of DNA contains a wild-type lacI+ gene. It encodes a wild-type trans-acting lac repressor that can bind to the chromosomal operator of the lac operon and restore wild-type regulation.

An E. coli strain of chromosomal genotype lacI lacP+ lacO+ lacZ+ lacY+ constitutively expresses the genes of the lac operon. The strain is converted to wild-type lac operon regulation by the addition of an extra piece of DNA. What gene or genes are contained on this extra DNA that explain this conversion to wild type? Include an explanation of cis- or trans-acting factors and how they work.

repressor protein

An example of a gene product encoded by a regulatory gene is beta-galactosidase enzyme. allolactose. repressor protein. an operator. a terminator.

negative repressible.

An operon is controlled by a repressor. When the repressor binds to a small molecule, it binds to DNA near the operon. The operon is constitutively expressed if a mutation prevents the repressor from binding to the small molecule. The type of control illustrated is negative inducible. negative repressible. positive inducible. positive repressible. catabolite repression.

negative inducible.

An operon is controlled by a repressor. When the repressor binds to a small molecule, it is released from binding to DNA near the operon. The operon is never expressed if a mutation prevents the repressor from binding to the small molecule. The type of control illustrated is negative inducible. negative repressible. positive inducible. positive repressible. attenuation.

A mutation (cis acting) in the promoter sequence of lacI could abolish lacI RNA and protein synthesis. In the absence of lacI protein, lac operon will be constitutively expressed.

Aside from the case of lacOc, where the operator is unable to bind to the repressor protein, briefly describe another mutant that could have caused constitutive expression of lac operon.

Sim: Both bind operator sequences in DNA Diff: when lac repressor binds the inducer, it cannot bind to DNA, but top does

Describe one similarity and one difference in how the trp and lac repressor proteins function.

positive inducible.

E. coli lac operon control by CAP is negative inducible. negative repressible. positive inducible. positive repressible.

negative inducible.

E. coli lac operon control by lacI is negative inducible. negative repressible. positive inducible. positive repressible. attenuation.

First, energy conservation is the key aspect that drives catabolite repression. Given the availability of carbon sources, whichever carbon sources will result in the least amount of energy expended to obtain their stored energy as well as any carbon sources that will result in net positive energy will be used by the E. coli. Second, the genes that are essential to utilize glucose are not dependent on c-AMP-CAP complex to promote their transcription. This nondependency allows glucose to shut transcription of other operons that are required to utilize alternate carbon sources.

Explain why glucose-dependent catabolite repression in E.coli is important and how it is possible to achieve this repression without influencing glucose metabolism.

Constitutive attenuation of transcription

If a mutation prevents the formation of the antiterminator 2+3 loop in the trp operon, what would be the effect? Transcription only when tryptophan is absent Transcription only when tryptophan is present Constitutive attenuation of transcription Constitutive transcription No effect, as 2+3 loop has no function

In the artificial construct, the normal regulatory apparatus of the lac operon is completely absent. Instead, the lacZ and lacY genes are now associated with the regulatory proteins and sequences of the trp operon. Therefore, the level of ß-galactosidase activity will be entirely dependent on tryptophan levels and entirely independent of lactose levels. In the absence of tryptophan, the trp repressor protein will be unbound, and the anti-terminator loop will form in the 5′ UTR due to ribosome stalling. Therefore, expression will be high, and ß-galactosidase activity will be high. When tryptophan levels are high, tryptophan will bind to the trp repressor and act as a co- repressor. Therefore, expression will be low. In addition, high tryptophan levels lead to read-through in the 5′ UTR, formation of the attenuation loop, and termination of transcription. As a result, lacZ will not be transcribed, ß-galactosidase will not be generated, and activity levels will be low.

Imagine the following scenario: You take the regulatory region of the trp operon (including the promoter, operator, and 5′ UTR) and attach it upstream of the structural genes of the lac operon. You then introduce this artificial construct into a mutant in which the lac operon is completely nonfunctional. Indicate the level of ß-galactosidase activity in each of the following cases and explain why you expect that level of activity: No tryptophan, no lactose High tryptophan, high lactose

The regulator falls off the operator and structural genes get transcribed.

In the absence of tryptophan, what happens to the genes within the trp operon? The regulator without tryptophan-binding prevents the genes from being transcribed. The regulator falls off the operator and structural genes get transcribed. Lack of tryptophan increases the level of cAMP high, which leads to activation of CAP protein and gene expression. The active repressor binds to the operator and genes do not get transcribed. The active activator binds to the operator and transcription of structural gene takes place.

A partial diploid strain with the genotype lacI+ lacZ−/lacI− lacZ+ was central to their efforts. This strain functioned normally, synthesizing β-galactosidase only when lactose was present. In this strain, the functional lacZ+ gene was not physically linked to the functional lacI+ gene. Despite that, since a lacI+ gene could regulate a lacZ+ gene located on a different DNA molecule, they concluded that a functional lacZ+ can act in trans to regulate the lac operon that is present either in the same chromosome.

In the experiments described in the text, Jacob and Monad deciphered that the lacI+ gene product can function in trans and regulate the lac operon either on the plasmid or on the chromosome. What genotype of the partial diploid bacterial strain was key to their experiments, leading them to such a conclusion? Explain why this strain helped them to reach that conclusion.

the repressor-binding site overlaps the promoter site of the operon, allowing it to physically block the binding of RNA polymerase.

It is possible for a repressor to negatively regulate the expression of an operon because the repressor induces the expression of the inducer by binding to the promoter that comes before the inducer gene. one of the structural genes expressed in the operon negatively regulates the repressor. the repressor-binding site overlaps the promoter site of the operon, allowing it to physically block the binding of RNA polymerase. d. the repressor-binding site on the DNA overlaps with the translation start site, hence preventing the transcription. e. the repressor physically blocks where the activator should be binding on the region.

major grooves

Proteins with DNA binding motifs predominantly bind to the ____________ of DNA. major grooves minor grooves paired nitrogenous bases phosphate groups deoxyribose sugar

antisense RNA.

RNA molecules that are complementary to particular sequence on mRNA are called complementary RNA. sense RNA. antisense RNA. riboswitches. ribozymes.

.ribozymes

RNA-mediated repression is carried out by nonsense RNA. sense RNA. antisense RNA. riboswitches. ribozymes

Translation through the 5 UTR would no longer be dependent on the availability of the amino acid tryptophan. (However, it would now be dependent on levels of cysteine.) Regardless of levels of tryptophan, translation would continue through the 5 UTR, the attenuator hairpin would form, and the antiterminator loop would form. The entire trp operon would not be transcribed (unless levels of cysteine were low).

The 5 UTR of the trp operon RNA contains several UGG tryptophan codons. What would be the effect on transcription-attenuation gene regulation if the trp codons were converted to UGC cysteine codons?

The basal level of activity is 20 units of activity, but in the absence of the mannose substrate, activity is reduced four-fold, to 5 units. In the presence of mannose, the basal level of activity is doubled to 40 units. In the partial diploid, the repressed level of expression is 10 units (5 units × 2) because there are two copies of the operon, and the induced level is 80 units (40 units × 2). m1: In the haploid, there is neither repression nor activation of the basal level of activity, as indicated by the 20 units of activity both in the presence and absence of substrate. This phenotype is corrected in trans by a trans-acting factor from the F′. The manR regulatory protein is the only trans-acting factor described in the system. Because the partial diploid is phenotypically wild type, the added manR protein must be sufficient for normal activity. Therefore, m1 is a mutant of the manR gene, such that the gene product can neither activate nor repress expression, and introduction of a wild-type version restores both activation and repression. m2: In the haploid, repression is normal in the absence of mannose, but there is no activation beyond the basal level with the addition of mannose. In the partial diploid, the defect is not corrected in trans, such that the addition of mannose only allows for a basal 20 units of activity from the chromosomal operon, and the full 40 units of activity from the F′ operon. Because the defect is cis-acting, affecting the ability to activate transcription, the mutation is likely in the manI initiator element such that the R2 form of the regulatory protein cannot bind and increase transcription. m3: In the haploid, there is no repression in the absence of mannose, but activation remains normal. In the partial diploid, there is no correction of the defect as levels of uninduced activity appear to be the sum of the regulated F′ (5 units) and the unregulated chromosomal copy (20 units). This mutation is thus in a cis-acting element affecting the ability to repress transcription. m3 is therefore a mutation in the manO operator site.

The bacterium, Bacillus subtilis, can grow on minimal media with a variety of sugars as carbon source. One such sugar is mannose, metabolized by the products of the man operon. Expression of the operon is controlled by a regulatory protein encoded in a separate gene, manR. Depending on conditions the regulatory protein may bind at one of two sites in the operon, as follows:

It is most likely due to the fact that the attenuation is the evolutionary relic, which by accident has remained.

The trp operon is known to operate by both negative repressible regulation of operator and attenuation. Which of the following does NOT support the reason as to why dual control exists to regulate the operon? The repression alone is never complete, and some transcription can be initiated. Combined mechanism provides a much finer tuning of tryptophan synthesis regulation. c. Attenuation and repression allow the cell to more sensitively respond to the tryptophan level. It is most likely due to the fact that the attenuation is the evolutionary relic, which by accident has remained. Repression responds to the cellular levels of tryptophan, while attenuation responds to the number of tRNA charged with tryptophan.

(1)Induction: Stimulates expression of a gene in response to a specific substrate.Repression: Prevents expression of a gene until a specific substrate is present.(2)Induction: Transcription is normally off and is stimulated in response to a specific substrate. Repression: Transcription is normally on and is shut off in response to a specific product.

What is the difference between a transcription regulation system that uses induction and a system that uses repression?

It activates an activator protein.

What is the function of cAMP in regulation of the lac operon? It activates a repressor protein. It activates an activator protein. It inactivates a repressor protein. It inactivates an activator protein. It causes attenuation.

The repressor would not be bound

Where would the lac repressor be bound in a (nonmutant) E. coli cell that is growing in low glucose and high lactose? (I = lac repressor gene; Z, Y, A = lac operon structural genes; P = lac promoter; O = lac operator) P O PandO I, P, O The repressor would not be bound.

For a gene under negative repressible control, a small molecule is required to prevent the gene's repressor from binding to DNA.

Which of the following statements about gene regulation concerning operon is INCORRECT? A repressible gene is controlled by a regulatory protein that inhibits transcription. For a gene under negative repressible control, a small molecule is required to prevent the gene's repressor from binding to DNA. For a gene under positive repressible control, the normal state is transcription of a gene, stimulated by a transcriptional activator. A regulator gene has its own promoter and is transcribed into an independent mRNA. Presence of operon where genes of related functions are clustered is common in bacteria, but not in eukaryotes.

Alternation of chromatin structure

Which of the following types of eukaryotic gene regulation is at the level of DNA? Alternation of chromatin structure mRNA processing RNA interference mRNA stability Post-translational modification

When glucose and lactose are present, E. coli prefer to use glucose. When lactose is high, allolactose binds the lac repressor, removing it from the operator and allowing transcription of the lac operon. However, when glucose is high, cAMP is low, so CAP does not activate transcription of the lac operon to its highest levels. As glucose is used up, shown by the dotted line going down, cAMP levels increase, cAMP binds CAP, and transcription of the lac operon is activated to its highest levels.

The dotted line in the following graph shows levels of glucose in a culture of wild-type E. coli grown in medium that initially contains both glucose and lactose. The solid line shows levels of transcription of the lac operon. Describe what is happening to the culture and the lac operon, referring to the lac repressor, allolactose, cAMP, and CAP (catabolite activator protein).

The nucleotide changes could change two aspects of the riboswitch: 1) they could disrupt the secondary structure of the mRNA, thereby preventing proper formation of a terminator structure, and causing constitutive transcription regardless of whether lysine is present or not; or 2) they could prevent binding of lysine (or another small regulatory molecule) to the mRNA, again preventing terminator formation and allowing constitutive transcription.

The lysC gene in Bacillus subtilis encodes the first enzyme in the metabolic pathway that generates lysine from its precursor molecule. In the presence of high concentrations of lysine, lysC expression is reduced. Researchers have generated mutant bacteria that produce the LysC enzyme constitutively, even in the presence of lysine. A mutant identified in this manner had two nucleotide changes in the 5′ UTR of the lysC gene itself. These nucleotide changes are thought to affect the function of a riboswitch regulating transcription of lysC. Describe two possible ways in which the mutations might affect the function of this riboswitch.

The repressor regulator protein binds to the operator and prevents the transcription of the structural gene.

What would happen to the lac operon in the absence of allolactose? The structural genes within the lac operon the will be constitutively transcribed. The activator protein will be bound to the operator, which will turn on the structural gene behind it. The repressor regulator protein binds to the operator and prevents the transcription of the structural gene. The catabolite activator protein becomes inactivated and no transcription occurs. The cAMP level rises in the absence of allolactose, which in turn inactivates the transcription.

The structural gene will be constitutively expressed due to the lack of negative inducible control.

When a structural gene is under negative inducible control, what would be the result of a mutation that eliminates the repressor protein? a. The structural gene will be constitutively expressed due to the lack of negative inducible control. The transcription of the structural gene will not be affected, as a repressor is not required. The mutation will lead to activation of an activator upon the lack of a repressor protein, which will allow the transcription to continue. As the transcription will require a repressor protein, the transcription will be turned off. More cAMP will be produced in a cell to compensate for the lack of a repressor protein.

. As the transcription will require an activator protein, the transcription will be turned off.

When a structural gene is under positive inducible control, what would be the result of a mutation that eliminates the activator protein? The structural gene to be constitutively expressed due to the lack of inducible control. The transcription of structural gene will not be affected, as an activator is not required. The mutation will lead to activation of a repressor upon the lack of an activator protein, which will block transcription. d. As the transcription will require an activator protein, the transcription will be turned off. e. More cAMP will be produced in a cell to compensate for the lack of an activator protein.

Homeodomain

Which of the following DNA binding motifs are composed of three alpha helices? a. Zinc-fingerb. Leucine-zipper c. Homeodomain d. Helix-turn-helix e. Helix-loop-helix

Riboswitches are typically found in the 3′ UTR of the mRNA structure.

Which of the following facts about riboswitches is INCORRECT? Binding of certain molecules to the riboswitches results in the formation of specific secondary structures of mRNA. Certain molecules that bind to riboswitches may act as repressors or inducers of transcription. Riboswitches are only found in bacterial cells but not in archaeal, fungal, or plant cells. Riboswitches are typically found in the 3′ UTR of the mRNA structure. The secondary structure that forms riboswitches typically contains a base stem and several branching hairpins.

The regulation of gene expression is critical for the control of life processes in all organisms.

Which of the following statement about regulation of gene expression is correct? An inducible gene is transcribed when a specific substance is absent. A gene is any DNA sequence that is transcribed into an mRNA molecule only. All genes are transcribed at all times as long as they have a functional promoter. The regulation of gene expression is the same in both eukaryotes and prokaryotes. The regulation of gene expression is critical for the control of life processes in all organisms.

Once bound, most of DNA binding proteins remain on DNA permanently.

Which of the following statements about DNA binding protein is NOT true? Specific amino acids within the motif form hydrogen bonds with DNA. These proteins can affect the expression of a gene. Most DNA binding proteins bind dynamically. Some of these proteins incorporate metal ion such as zinc. Once bound, most of DNA binding proteins remain on DNA permanently.

The following table shows several bacterial strain lac operon genotypes (some are partial diploids). Fill in the blanks in the "lactose absent" and "lactose present" columns in this table. (+) means significant levels of active ß-galactosidase enzyme can be detected. (-) means no significant levels of active ß-galactosidase enzyme. The first line is filled in for reference. If lactose is absent and glucose is present in the culture medium of strain 4, will significant levels of active ß-galactosidase enzyme be detected? Explain. Using your answers in the previous table, explain the dominance relationships among the three lacI alleles (lacI+, lacI−, lacIs).

Yes, despite the fact that the super-repressor allele is present (lacIs), it cannot bind to the operator due to the lacOc mutation at the operator—this allows for constitutive ß- galactosidase enzyme expression (expression whether lactose is present of absent). If glucose is present, the CAP protein will NOT be active (cAMP levels will be low). The combination of lack of repression and lack of activation by CAP will most likely result in transcription of the operon and production of active ß-galactosidase enzyme. lacIs is (trans) dominant to both lacI+ and lacI−; lacI+ is (trans) dominant to lacI−. When both lacI+ and lacI− are present in the cell, we see the lacI+ phenotype (see strain 5). When lacIs is present in the cell, we see the lacIs phenotype (see strain 6). Section: 16.2

Because only the bacterial chromosome has a functional lacY gene and only the F′ has a functional lacZ gene, we can think of permease activity (from lacY+) and ß- galactosidase activity (from lacZ+) as reporters for lac operon expression from the bacterial chromosome and the F′, respectively. That is, permease activity indicates whether the operon on the bacterial chromosome is regulated, constitutive, or uninducible; and ß-galactosidase activity indicates whether the operon on the F′ is regulated, constitutive, or uninducible. Lac1- is a dominant, cis-acting mutation. It is dominant because permease activity is still constitutive in the partial diploid with one wild-type allele (the F′ should contain a Lac1+ allele) and the chromosomal Lac1- allele. Lac1- is cis-acting because it can only confer constitutive expression on the reporter in cis (on the same piece of DNA, lacY+), while the reporter in trans (on a physically separate piece of DNA, lacZ+) is regulated appropriately. Lac2- is a dominant, trans-acting mutation. It is dominant because permease activity is still constitutive in the partial diploid with the wild-type allele on the F′ and the chromosomal Lac2- mutation. Lac2- is trans-acting because it can confer constitutive expression on both the reporter in cis (lacY+) and the reporter in trans (lacZ+). Lac1- best fits the properties of lacOC mutations (cis-acting dominant). A mutation in the operator sequence prevents binding of the LacI repressor, causing constitutive expression, but only for genes on the same chromosome. A trans-acting factor is usually a protein, whereas a cis-acting factor is usually a DNA sequence. Therefore, the Lac2- mutation appears to be a mutation affecting a protein. Besides the structural genes, the only protein component of the lac operon is the LacI repressor protein. However, most of the time, mutations in lacI are recessive, because a functional copy of lacI on an F′ can substitute for a lacI mutation in the bacterial chromosome. For this allele to be dominant, we have to propose that the mutant form of LacI protein generated by the Lac2- mutation has to actually interfere with the function of the normal LacI protein produced from the F′. A mutant allele that can interfere with the function of a normal allele is typically referred to as a dominant negative, and mutations of this kind that have been identified in the lac operon are known as lacId mutations.

You have isolated two mutations linked to the lac operon, which you designate Lac1- and Lac2- that cause constitutive expression of the operon. You construct strains carrying a lac operon with a mutant lacY gene on an F′. You test both ß-galactosidase activity and Lac