Genetics ch.14
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. 3. trp operon: The binding of tryptophan to trp repressor causes it to bind to the operator site and inhibits transcription.
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 the 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.
As described in Chapter 13, enzymes known as aminoacyl-tRNA synthetases are responsible for attaching amino acids to tRNAs. Let's suppose that in a mutant bacterium tryptophanyl-tRNA synthetase has a reduced ability to attach tryptophan to tRNA: its activity is only 10% of that found in a normal bacterium. How would attenuation of the trp operon be affected? Would the operon be more or less likely to be attenuated? Explain your answer.
A defective tryptophanyl-tRNA synthetase would make attenuation less likely. This is because the bacterial cell would have a lower amount of charged tRNATrp. 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 cell was high. When the ribosome stalls at these tryptophan codons, this prevents attenuation.
Some mutations have a cis-effect, whereas others have a trans-effect. Explain the molecular differences between cis- and trans-mutations. 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.
Would a mutation that inactivated 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.
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 will 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 to parts A-C that list more than one mechanism, which mechanism will 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.
As shown 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 each of the following sets of 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 2-3 stem-loop will form. B. Attenuation will occur because a 2-3 stem-loop cannot form, so a 3-4 stem-loop will form. C. Attenuation will not occur because a 3-4 stem-loop cannot form. D. Attenuation will not occur because a 3-4 stem-loop cannot form.
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 lac repressor from binding to it B. A mutation in the lacI gene that prevents lac repressor from binding to DNA C. A mutation in the trpL gene that prevents attenuation
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. C. Cis-effect. It would affect only the genes that are in the adjacent operon.
In the lac operon, how would gene expression be affected if each 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.
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 each of the following terms apply to a regulatory protein, a segment of DNA, or a small effector molecule? A. Repressor B. Inducer C. Operator site D. Corepressor E. Activator F. Attenuator G. Inhibitor
A. Regulatory protein B. Effector molecule C. DNA segment D. Effector molecule E. Regulatory protein F. DNA segment G. Effector molecule
What is antisense RNA? How does it affect the translation of a complementary mRNA?
Antisense RNA is RNA that is complementary to a functional RNA such as mRNA. The binding of antisense RNA to mRNA inhibits translation.
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 that 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 trpL region plays a key role in the attenuation mechanism.
An operon is repressible—a small effector molecule turns off its transcription. Which combination(s) of small effector molecule and regulatory protein could be involved in this process? 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 cases, the presence of the small effector molecule will turn off transcription. In contrast, the presence of an inducer turns on transcription.
What is diauxic growth? Explain the roles of cAMP and CAP 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 type (such as lactose). This process is governed 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.
Mutations in tRNA genes can create tRNAs that recognize stop codons. Because stop codons are sometimes called nonsense codons, these types of mutations that affect tRNAs are called nonsense suppressors. For example, a normal tRNAGly 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 tRNAGly could change the anticodon to ACU. This mutant tRNAGly 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 located after region 1.
If you look very carefully at the RNA sequence in Figure 14.12, you will notice that a UAA codon is found just past region 2. Therefore, in this mutant strain, the UGA stop codon at the end of region 1 could be read by the mutant tRNAGly 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 3, and therefore the terminator 3-4 stem-loop would not form. According to this scenario, attenuation could not occur. However, you should also keep in mind the issue of timing. The ribosome would have to be really close to RNA polymerase to prevent attenuation in this nonsense suppressor strain. It is possible that the 3-4 stem-loop might form before the ribosome reaches the UAA stop codon just past region 2. Therefore, attenuation might occur anyway because the 3-4 stem-loop might form before the ribosome reaches the UAA stop codon.
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 a gene only when the gene product is needed. For example, a bacterium will express only the genes that are necessary for lactose metabolism only when the 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 that are required for tryptophan biosynthesis are repressed.
If a gene is repressible and under positive control, what kind of effector molecule and regulatory protein are involved in its regulation? 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.
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 stopping the process a later stage, such as elongation or termination.
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 species of bacteria can synthesize the amino acid histidine, so they do not require histidine in their 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 medium, 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 is consistent with this observation?
One possible mechanism is that histidine acts as corepressor that shuts down the transcription of the histidine synthetase gene. A second mechanism might be that histidine acts as an inhibitor via feedback inhibition. A third possibility is that histidine inhibits the ability of the mRNA that encodes histidine synthetase to be translated. Perhaps histidine 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.
The combination of a 3-4 stem-loop and a U-rich attenuator in the trp operon (see Figure 14.12) is an example of a ρ-independent terminator. 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 C 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 C. Remember that GC base pairs have three hydrogen bonds and are more stable than AU base pairs, which have only two hydrogen bonds.
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.
How are the actions of 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.
