Genetics Chapter 14

How might antisense RNA affect translation? A) It can interfere with ribosome binding by blocking binding sites. B) It can enhance ribosome binding by modifying binding sites. C) It can prevent identification of the start codon by the ribosome. D) It can interfere with the normal secondary structure of the mRNA molecule.

A

Which of the following genes is not a structural gene of the lac operon? A) lacA B) lacI C) lacY D) lacZ

B

Describe enzyme adaptation.

Production of the enzyme only occurs when substrate becomes available, and stops when the supply of substrate is exhausted.

C9. 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.

A gene which is usually turned down but can be activated is under negative control. A) True B) False

A

Catabolite repression requires an inducer and an activator protein. A) True B) False

A

The most convincing evidence that lacI is a diffusible protein is: A) A merozygote with only one functional lacI gene is able to regulate both copies of the lac operon. B) A merozygote with only one functional lacO is unable to regulate both copies of the lac operon. C) The lacI gene is located very close to the lac operon. D) Mutants lacking lacI function do not respond to changes in lactose concentration. E) All of the above.

A

Which of the following would you expect to find in an inducible system? A) A repressor protein, which is bound to DNA in absence of any other factor. B) A repressor protein, which is bound to DNA in the presence of a corepressor. C) An activator protein, which is bound to DNA in absence of any other factor. D) An activator protein, which is bound to DNA only in the absence of an inhibitor.

A

Why does the ribosome stall on region 1 during attenuation? A) Translation of this region requires tRNAtrp. B) High levels of tryptophan interfere with ribosome function. C) Low levels of tryptophan lead to intrinsic termination. D) Binding of the repressor protein prevents further translation.

A

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

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

C12. 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.

C8. 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.

C4. 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 E. Activator B. Inducer F. Attenuator C. Operator site G. Inhibitor D. Corepressor

A. Regulatory protein E. Regulatory protein B. Effector molecule F. DNA segment C. DNA segment G. Effector molecule D. Effector molecule

Give two lines of evidence that indicate the lac operon is inducible

Addition of lactose to the culture medium dramatically increases the levels of lactose utilization enzyme expression; removal of lactose leads to an abrupt termination of expression of these enzymes; mutations that allow constitutive expression of these enzymes exist; other answers are possible.

C19. What is antisense RNA? How does it affect the translation of a complementary mRNA?

Antisense RNA is RNA that is complementary to a fundamental RNA such as mRNA. The binding of antisense RNA to mRNA inhibits translation.

C13. What is meant by the term attenuation? Is it an example of gene regulation at the level of transcription or translation? Explain your answer.

Attenuation means the transcription is ended before it has reached the end of an operon. Because it causes an end to transcription it is a form of transcriptional regulation even though the translation of the TRP L region plays a key role in the attenuation mechanism

A translational regulatory protein: A) Binds to DNA and prevents translation. B) Binds to mRNA and prevents translation. C) Binds to rRNA and prevents translation. D) Binds to tRNA and prevents translation.

B

All of the genes involved in functioning of the lac operon are inducible. A) True B) False

B

The inducer for the lac operon is: A) lactose B) allolactose C) β-galactosidase D) galactose

B

The lac repressor protein can repress the operon by binding to any one of the three binding sites in the operator. A) True B) False

B

Transcriptional regulation: A) Is highly efficient at completely preventing transcription. B) Allows the cell to only produce proteins that are needed at the time. C) Can be induced by a repressor protein. D) All of these. E) None of these.

B

C5. An operon is repressible—a small effector molecule turns off transcription. Which combinations of small effector molecules and regulatory proteins could be involved? A. An inducer plus a repressor B. A corepressor plus a repressor C. An inhibitor plus an activator D. An inducer plus an activator

B and C are correct. In both of these cases, the presence of the small effector molecule will turn off transcription. In contrast, the presence of an inducer turns on transcription.

What effect would you expect if gene expression of the lac operon were completely repressed? A) The cell would be more efficient without "wasting" the energy required for the low level of lacZ, lacY, and lacA gene expression. B) Allolactose would accumulate within the cell and become toxic. C) Lactose would not be converted into the inducer and the operon could not be induced. D) All of these. E) A and B only.

C

Which of the following elements, when nonfunctional, would lead to a reduction in the total amount of functional lacZ produced by a bacterial cell? A) The operator. B) The lacI gene. C) The CAP site. D) The lacA gene. E) The lacY gene.

C

Genes within an operon: A) Tend to be regulated by a common regulatory mechanism. B) Are generally involved in the same biochemical pathway. C) Are expressed as a polycistronic RNA. D) All of the above. E) None of the above.

D

Which of the following would be an example of enzyme adaptation? A) Enzymes required for glucose utilization are constitutively expressed. B) Addition of phosphate groups to a protein can activate it. C) Certain enzymes are made up of multiple subunits. D) Enzymes for tryptophan synthesis are not made in the presence of tryptophan.

D

C10. 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.

An allosteric enzyme: A) Has two different binding sites. B) Is regulated by binding of a regulatory molecule somewhere other than the active site. C) May be regulated by feedback inhibition. D) Is an example of posttranslational regulation. E) All of these.

E

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.

C3. 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.

In this case, 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.

C18. Translational control is usually aimed at preventing the initiation of translation. With regard to cellular efficiency, why do you think this is the case?

It takes a lot of cellular energy to translate mRNA into a protein. A cell wastes less energy if it prevents the initiation of translation rather than a later stage such as elongation or termination.

Which point of regulation would a bacterium be most likely to use if an important goal was quick production of functional protein?

Posttranslation.

Would you expect repression or attenuation of the trp operon to be a more efficient method?

Repression, because attenuation requires transcription and translation of the trpL sequence.

List two examples of metabolic proteins regulated by bacteria.

Structural genes of the lac, trp, or ara operons; other answers are possible.

C7. What is enzyme adaptation? From a genetic point of view, how does it occur?

Th term enzyme adaptation means that a particular enzyme is made only when a cell is exposed to the substrate for he 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.

C16. The 3-4 stem-loop and U-rich attenuator found in the trp operon (see Figure 14.12) is an example of ρ-independent termination. The function of ρ-independent terminators is described in Chapter 12. Would you expect attenuation to occur if the tryptophan levels were high and mutations changed the attenuator sequence from UUUUUUUU to UGGUUGUC? Explain why or why not.

The addition of Gs and Cs into the U-rich sequence would prevent attenuation. The U-rich sequence promotes the dissociation of the mRNA from the DNA, when the terminator stem-loop forms. This causes RNA polymerase to dissociate from the DNA and thereby causes transcriptional termination. The UGGUUGUC sequence would probably not dissociate because of the Gs and Cs. Remember that GC base pairs have three hydrogen bonds and are more stable than AU base pairs, which have only two hydrogen bonds.

C22 In what ways are the actions of the lac repressor and trp repressor similar and how are they different with regard to their binding to operator sites, their effects on transcription, and the influences of small effector molecules?

The two proteins are similar in that both bind to a segment of DNA and repress transcription. They are different in three ways. (1) They recognize different effector molecules (i.e., the lac repressor recognizes allolactose, and the trp repressor recognizes tryptophan). (2) Allolactose causes the lac repressor to release from the operator, while tryptophan causes the trp repressor to bind to its operator. (3) The sequences of the operator sites that these two proteins recognize are different from each other. Otherwise, the lac repressor could bind to the trp operator, and the trp repressor could bind to the lac operator.

How many ribosomes are required for synthesis of the trp structural genes?

Two, one to prevent attenuation and one to translate the structural genes.

What advantage does diauxic growth present for a bacterium?

Utilization of glucose with constitutively active enzymes is preferred over utilization of other sugars which require specialized enzymes. This is expected to be more efficient for the bacterium.

C21. Using three examples, describe how allosteric sites are important in the function of genetic regulatory proteins.

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

C6. 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.

C23. Transcriptional repressor proteins (e.g., lac repressor), antisense RNA, and feedback inhibition are three different mechanisms that turn off the expression of genes and gene products. Which of these three mechanisms would be most effective in each of the following situations? A. Shutting down the synthesis of a polypeptide B. Shutting down the synthesis of mRNA C. Shutting off the function of a protein For your answers in parts A-C that have more than one mechanism, which mechanism would be the fastest or the most efficient?

A. Antisense RNA or a translational repressor would shut down protein synthesis the fastest. A transcriptional repressor would also shut down the synthesis of mRNA, so it would eventually shut down protein synthesis once all of the preexisting mRNA had been degraded. Feedback inhibition would have no effect on protein synthesis. B. Only a transcriptional repressor protein would shut down the synthesis of mRNA C. Feedback inhibition is the fastest way to shut down the function of a protein. Antisense RNA and transcriptional repressors eventually prevent protein function once all of the preexisting mRNA and protein have been degraded.

C15. As described in Chapter 13, enzymes known as aminoacyl-tRNA synthetases are responsible for attaching amino acids to tRNAs. Let's suppose that tryptophanyl-tRNA synthetase was partially defective at attaching tryptophan to tRNA; its activity was only 10% of that found in a normal bacterium. How would attenuation of the trp operon be affected? Would it be more or less likely to be attenuated? Explain your answer.

Affective tryptophanyl tRNA Synthetase would make attenuation less likely. This is because the bacterial cell would have a lower amount of charged tRNA. Therefore it would be more likely for the ribosome to stall at the tryptophan codons found within the trpL gene even if the concentration of tryptophan amino acids in the cells high. When the ribosome stalls at these tryptophan codons this prevents attenuation

What is the main difference between an activator protein and an inducer?

An activator protein is a protein that can bind directly to DNA. An inducer is a small molecule which either binds a regulatory protein and prevents it from binding DNA or binds to an activator protein and allows it to bind DNA.

If regulation of a gene involves a repressor protein, it is an inducible gene. A) True B) False

B

C14. As described in Figure 14.12, four regions within the trpL mRNA can form stem-loops. Let's suppose that mutations have been previously identified that prevent the ability of a particular region to form a stem-loop with a complementary region. For example, a region 1 mutant cannot form a 1-2 stem-loop, but it can still form a 2-3 or 3-4 stem-loop. Likewise, a region 4 mutant can form a 1-2 or 2-3 stem-loop but not a 3-4 stem-loop. Under the following conditions, would attenuation occur? A. Region 1 is mutant, tryptophan is high, and translation is not occurring. B. Region 2 is mutant, tryptophan is low, and translation is occurring. C. Region 3 is mutant, tryptophan is high, and translation is not occurring. D. Region 4 is mutant, tryptophan is low, and translation is not occurring.

A. Attenuation will not occur because loop 2-3 will form. B. Attenuation will occur because 2-3 cannot form, so 3-4 will form. C. Attenuation will not occur because 3-4 cannot form. D. Attenuation will not occur because 3-4 cannot form.

C11. 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 C. A mutation in trpL that prevents attenuation

A. cis-effect. It would affect only the genes that are in he adjacent operon. B. trans-effect. This is a mutation that affects a protein that can move throughout the cell C. cis-effect. It would affect only the genes that are in he adjacent operon.

Attenuation of the trp operon: A) Occurs when transcription is complete before translation begins. B) Is mediated by the trp repressor protein. C) Occurs in the presence of high levels of tryptophan. D) All of the above. E) None of the above.

C

What is the best explanation for the stem loop arrangement seen in the absence of translation of the trpL gene? A) Region 4 binds to region 3 before region 2 has been transcribed. B) Region 1 is unable to bind to region 3 or 4. C) The 1:2 stem loop has more base pairs than a 2:3 stem loop. D) The ribosome prevents region 1 from binding to any other region.

C

C17. Mutations in tRNA genes can create tRNAs that recognize stop codons. Because stop codons are sometimes called nonsense codons, the types of mutations that affect tRNAs are called nonsense suppressors. For example, a normal tRNA Gly has an anticodon sequence CCU that recognizes a glycine codon in mRNA (GGA) and puts in a glycine during translation. However, a mutation in the gene that encodes tRNA Gly could change the anticodon to ACU. This mutant tRNA Gly would still carry glycine, but it would recognize the stop codon UGA. Would this mutation affect attenuation of the trp operon? Explain why or why not. Note: To answer this question, you need to look carefully at Figure 14.12 and see if you can identify any stop codons that may exist beyond the UGA stop codon that is found after region 1.

If you look very carefully at the RNA sequence in figure 14.12 you'll notice that a UAA codon is found just past region two. Therefore in this mutant strain the UGA stop codon at the end of region one could be read by the mutant tRNA and then the ribosome would stop at the UAA codon just past region 2. If the ribosome paused here it would probably cover up a portion of region three and therefore the terminators 3- 4 Stem Loop would not form. According to the scenario attenuation cannot occur. However we should also keep in mind that issue of timing. The ribosome would have to be really close to RNA polymerize to prevent attenuation in this nonsense repressor strain. It is possible that the 3 - 4 Stem Loop wait form before the ribosome reaches the UAA stop codon just passed region two. Therefore, attenuation might occur anyway because the 3-4 stem-loop might form before the ribosome reaches the UAA stop codon.

C20. A species of bacteria can synthesize the amino acid histidine so it does not require histidine in its growth medium. A key enzyme, which we will call histidine synthetase, is necessary for histidine biosynthesis. When these bacteria are given histidine in their growth media, they stop synthesizing histidine intracellularly. Based on this observation alone, propose three different regulatory mechanisms to explain why histidine biosynthesis ceases when histidine is in the growth medium. To explore this phenomenon further, you measure the amount of intracellular histidine synthetase protein when cells are grown in the presence and absence of histidine. In both conditions, the amount of this protein is identical. Which mechanism of regulation would be consistent with this observation?

One mechanism is that histidine could act as corepressor that shuts down the transcription of the histidine synthetase gene. A second mechanism would be that histidine could act as an inhibitor via feedback inhibition. A third possibility is that histidine inhibits the ability of the mRNA encoding histidine synthetase to be translated. Perhaps it induces a gene that encodes an antisense RNA. If the amount of histidine synthetase protein was identical in the presence and absence of extracellular histidine, a feedback inhibition mechanism is favored, because this affects only the activity of the histidine synthetase enzyme, not the amount of the enzyme. The other two mechanisms would diminish the amount of this protein.