題目 1 · free-response
9 分In eukaryotic microorganisms, cellular zinc homeostasis is maintained through the regulated expression of specific transport proteins. When extracellular zinc (\(\text{Zn}^{2+}\)) concentrations are low, an intracellular signaling pathway activates the transcription factor ZapA. Activation occurs when the protein kinase KinZ phosphorylates ZapA, inducing a conformational change that allows it to bind to the promoter region of the ZUP1 gene and stimulate transcription. ZUP1 encodes a high-affinity plasma membrane zinc transporter, Zup1. When extracellular \(\text{Zn}^{2+}\) is abundant, zinc directly binds to ZapA, promoting its interaction with a co-repressor complex comprising Cbp1 and Cbp2, which prevents ZapA from activating transcription of ZUP1.
To investigate the individual contributions of these regulatory components, researchers generated two mutant strains from a wild-type yeast strain: a mutant lacking functional KinZ (\(kinZ^{mt}\)) and a mutant lacking functional Cbp1 (\(cbp1^{mt}\)). Cells of the wild-type and mutant strains were cultured in media containing either high or low levels of \(\text{Zn}^{2+}\). The researchers measured Zup1 transport activity and quantified relative ZUP1 mRNA levels relative to wild-type cells grown in high \(\text{Zn}^{2+}\) (Table 1).
**TABLE 1. Zup1 TRANSPORT ACTIVITY AND RELATIVE ZUP1 mRNA LEVELS IN WILD-TYPE AND MUTANT YEAST STRAINS UNDER HIGH- AND LOW-ZINC CONDITIONS**
| Yeast Strain | Mutation | Zup1 Activity in High-\(\text{Zn}^{2+}\) (\(\text{nmol}/\text{min}/\text{mg}\) protein \(\pm 2\text{SE}_{\bar{x}}\)) | Zup1 Activity in Low-\(\text{Zn}^{2+}\) (\(\text{nmol}/\text{min}/\text{mg}\) protein \(\pm 2\text{SE}_{\bar{x}}\)) | Relative ZUP1 mRNA in High-\(\text{Zn}^{2+}\) (\(\pm 2\text{SE}_{\bar{x}}\)) | Relative ZUP1 mRNA in Low-\(\text{Zn}^{2+}\) (\(\pm 2\text{SE}_{\bar{x}}\)) |
| :--- | :--- | :--- | :--- | :--- | :--- |
| Wild-type | None | \(1.2 \pm 0.2\) | \(25.2 \pm 1.4\) | \(1.0 \pm 0.1\) | \(18.0 \pm 1.5\) |
| \(kinZ^{mt}\) | Nonfunctional KinZ kinase | \(1.1 \pm 0.2\) | \(1.4 \pm 0.3\) | \(0.9 \pm 0.2\) | \(1.2 \pm 0.3\) |
| \(cbp1^{mt}\) | Nonfunctional Cbp1 co-repressor | \(23.8 \pm 1.6\) | \(24.9 \pm 1.5\) | \(17.5 \pm 1.3\) | \(18.2 \pm 1.4\) |
(a) Describe the mechanism by which the addition of a negatively charged phosphate group by a kinase can alter the activity of a transcription factor such as ZapA. Explain how a signaling cascade can amplify a cellular signal during signal transduction.
(b) Based on Table 1, identify ONE dependent variable measured in the researchers' experiment. Justify the researchers' decision to use the wild-type yeast strain as the genetic background for engineering the mutant strains. Justify the researchers' testing of single-gene mutant strains rather than strains with multiple simultaneous mutations.
(c) Based on the data in Table 1, identify the yeast strain and growth condition that resulted in the lowest relative ZUP1 mRNA level. Calculate the percent change in Zup1 transport activity in wild-type yeast cells grown in low-\(\text{Zn}^{2+}\) compared to wild-type yeast cells grown in high-\(\text{Zn}^{2+}\).
(d) In a subsequent experiment, researchers generated a yeast strain with a loss-of-function mutation in the gene encoding Cbp2. Predict the effect of this mutation on ZUP1 mRNA expression when the mutant yeast is grown in a high-\(\text{Zn}^{2+}\) environment. Provide reasoning to justify your prediction.
To investigate the individual contributions of these regulatory components, researchers generated two mutant strains from a wild-type yeast strain: a mutant lacking functional KinZ (\(kinZ^{mt}\)) and a mutant lacking functional Cbp1 (\(cbp1^{mt}\)). Cells of the wild-type and mutant strains were cultured in media containing either high or low levels of \(\text{Zn}^{2+}\). The researchers measured Zup1 transport activity and quantified relative ZUP1 mRNA levels relative to wild-type cells grown in high \(\text{Zn}^{2+}\) (Table 1).
**TABLE 1. Zup1 TRANSPORT ACTIVITY AND RELATIVE ZUP1 mRNA LEVELS IN WILD-TYPE AND MUTANT YEAST STRAINS UNDER HIGH- AND LOW-ZINC CONDITIONS**
| Yeast Strain | Mutation | Zup1 Activity in High-\(\text{Zn}^{2+}\) (\(\text{nmol}/\text{min}/\text{mg}\) protein \(\pm 2\text{SE}_{\bar{x}}\)) | Zup1 Activity in Low-\(\text{Zn}^{2+}\) (\(\text{nmol}/\text{min}/\text{mg}\) protein \(\pm 2\text{SE}_{\bar{x}}\)) | Relative ZUP1 mRNA in High-\(\text{Zn}^{2+}\) (\(\pm 2\text{SE}_{\bar{x}}\)) | Relative ZUP1 mRNA in Low-\(\text{Zn}^{2+}\) (\(\pm 2\text{SE}_{\bar{x}}\)) |
| :--- | :--- | :--- | :--- | :--- | :--- |
| Wild-type | None | \(1.2 \pm 0.2\) | \(25.2 \pm 1.4\) | \(1.0 \pm 0.1\) | \(18.0 \pm 1.5\) |
| \(kinZ^{mt}\) | Nonfunctional KinZ kinase | \(1.1 \pm 0.2\) | \(1.4 \pm 0.3\) | \(0.9 \pm 0.2\) | \(1.2 \pm 0.3\) |
| \(cbp1^{mt}\) | Nonfunctional Cbp1 co-repressor | \(23.8 \pm 1.6\) | \(24.9 \pm 1.5\) | \(17.5 \pm 1.3\) | \(18.2 \pm 1.4\) |
(a) Describe the mechanism by which the addition of a negatively charged phosphate group by a kinase can alter the activity of a transcription factor such as ZapA. Explain how a signaling cascade can amplify a cellular signal during signal transduction.
(b) Based on Table 1, identify ONE dependent variable measured in the researchers' experiment. Justify the researchers' decision to use the wild-type yeast strain as the genetic background for engineering the mutant strains. Justify the researchers' testing of single-gene mutant strains rather than strains with multiple simultaneous mutations.
(c) Based on the data in Table 1, identify the yeast strain and growth condition that resulted in the lowest relative ZUP1 mRNA level. Calculate the percent change in Zup1 transport activity in wild-type yeast cells grown in low-\(\text{Zn}^{2+}\) compared to wild-type yeast cells grown in high-\(\text{Zn}^{2+}\).
(d) In a subsequent experiment, researchers generated a yeast strain with a loss-of-function mutation in the gene encoding Cbp2. Predict the effect of this mutation on ZUP1 mRNA expression when the mutant yeast is grown in a high-\(\text{Zn}^{2+}\) environment. Provide reasoning to justify your prediction.
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解題
(a)
- Description: The addition of a negatively charged phosphate group introduces electrostatic interactions (such as repulsion or attraction with neighboring amino acid residues) that alter the tertiary/three-dimensional conformation of the transcription factor, exposing its DNA-binding domain or altering its affinity for target DNA sequences.
- Explanation: Signal amplification occurs because a single activated enzyme (such as an upstream protein kinase) can catalyze the modification/activation of many downstream target proteins or secondary messengers, multiplying the signal at each successive step in the pathway.
(b)
- Identification: Either "Zup1 transport activity" or "relative ZUP1 mRNA levels / expression".
- Justification (wild-type control): Using the isogenic wild-type background ensures that all strains are genetically identical except for the specific targeted mutation, guaranteeing that any observed phenotypic differences are directly attributable to the introduced mutation rather than background genetic variation.
- Justification (single mutations): Mutating only a single gene at a time allows researchers to isolate and determine the specific, independent function of each individual component in the regulatory pathway.
(c)
- Identification: \(kinZ^{mt}\) in a high-\(\text{Zn}^{2+}\) environment (relative mRNA level of \(0.9 \pm 0.2\)).
- Calculation:
\[\text{Percent Change} = \frac{\text{Value}_{\text{low}} - \text{Value}_{\text{high}}}{\text{Value}_{\text{high}}} \times 100\%\]
\[\text{Percent Change} = \frac{25.2 - 1.2}{1.2} \times 100\% = \frac{24.0}{1.2} \times 100\% = 2,000\%\]
*(An increase of \(2,000\%\))*
(d)
- Prediction: ZUP1 mRNA expression will be high/constitutively expressed (elevated compared to wild-type under high-\(\text{Zn}^{2+}\) conditions).
- Justification: Cbp2 is an essential subunit of the co-repressor complex that normally functions with Cbp1 to inhibit ZapA under high-\(\text{Zn}^{2+}\) conditions. Without functional Cbp2, the co-repressor complex cannot form or inhibit ZapA, leaving ZapA active and free to promote ZUP1 transcription even in the presence of high zinc.
- Description: The addition of a negatively charged phosphate group introduces electrostatic interactions (such as repulsion or attraction with neighboring amino acid residues) that alter the tertiary/three-dimensional conformation of the transcription factor, exposing its DNA-binding domain or altering its affinity for target DNA sequences.
- Explanation: Signal amplification occurs because a single activated enzyme (such as an upstream protein kinase) can catalyze the modification/activation of many downstream target proteins or secondary messengers, multiplying the signal at each successive step in the pathway.
(b)
- Identification: Either "Zup1 transport activity" or "relative ZUP1 mRNA levels / expression".
- Justification (wild-type control): Using the isogenic wild-type background ensures that all strains are genetically identical except for the specific targeted mutation, guaranteeing that any observed phenotypic differences are directly attributable to the introduced mutation rather than background genetic variation.
- Justification (single mutations): Mutating only a single gene at a time allows researchers to isolate and determine the specific, independent function of each individual component in the regulatory pathway.
(c)
- Identification: \(kinZ^{mt}\) in a high-\(\text{Zn}^{2+}\) environment (relative mRNA level of \(0.9 \pm 0.2\)).
- Calculation:
\[\text{Percent Change} = \frac{\text{Value}_{\text{low}} - \text{Value}_{\text{high}}}{\text{Value}_{\text{high}}} \times 100\%\]
\[\text{Percent Change} = \frac{25.2 - 1.2}{1.2} \times 100\% = \frac{24.0}{1.2} \times 100\% = 2,000\%\]
*(An increase of \(2,000\%\))*
(d)
- Prediction: ZUP1 mRNA expression will be high/constitutively expressed (elevated compared to wild-type under high-\(\text{Zn}^{2+}\) conditions).
- Justification: Cbp2 is an essential subunit of the co-repressor complex that normally functions with Cbp1 to inhibit ZapA under high-\(\text{Zn}^{2+}\) conditions. Without functional Cbp2, the co-repressor complex cannot form or inhibit ZapA, leaving ZapA active and free to promote ZUP1 transcription even in the presence of high zinc.
評分準則
Part (a): 2 points maximum
- 1 point for describing that phosphorylation changes the conformation/tertiary shape/charge distribution of the protein.
- 1 point for explaining that one activated enzyme/kinase can catalyze reactions on multiple downstream protein molecules, thereby multiplying the molecular signal.
Part (b): 3 points maximum
- 1 point for identifying a dependent variable:
- Accept one of: Zup1 (transport) activity OR (relative) ZUP1 mRNA levels.
- 1 point for justifying using the wild-type strain:
- Accept one of: Ensures differences are solely due to the introduced mutation / controls for background genetic differences.
- 1 point for justifying using single-gene mutations:
- Accept one of: Allows the effect of each individual component/gene to be evaluated independently / determines the specific role of each pathway component.
Part (c): 2 points maximum
- 1 point for identifying \(kinZ^{mt}\) yeast in a high-\(\text{Zn}^{2+}\) environment.
- 1 point for correctly calculating the percent change:
- Accept: \(2,000\%\) (or \(+2,000\%\)) based on \(\frac{25.2 - 1.2}{1.2} \times 100\%\) [also accept \(-95.2\%\) if comparing high relative to low: \(\frac{1.2 - 25.2}{25.2} \times 100\%\)].
Part (d): 2 points maximum
- 1 point for predicting that ZUP1 will be expressed / transcribed / mRNA levels will be high/elevated in the mutant strain in a high-\(\text{Zn}^{2+}\) environment.
- 1 point for providing reasoning that without functional Cbp2, the co-repressor complex fails to assemble/inhibit ZapA, allowing ZapA to bind the promoter and activate transcription.
- 1 point for describing that phosphorylation changes the conformation/tertiary shape/charge distribution of the protein.
- 1 point for explaining that one activated enzyme/kinase can catalyze reactions on multiple downstream protein molecules, thereby multiplying the molecular signal.
Part (b): 3 points maximum
- 1 point for identifying a dependent variable:
- Accept one of: Zup1 (transport) activity OR (relative) ZUP1 mRNA levels.
- 1 point for justifying using the wild-type strain:
- Accept one of: Ensures differences are solely due to the introduced mutation / controls for background genetic differences.
- 1 point for justifying using single-gene mutations:
- Accept one of: Allows the effect of each individual component/gene to be evaluated independently / determines the specific role of each pathway component.
Part (c): 2 points maximum
- 1 point for identifying \(kinZ^{mt}\) yeast in a high-\(\text{Zn}^{2+}\) environment.
- 1 point for correctly calculating the percent change:
- Accept: \(2,000\%\) (or \(+2,000\%\)) based on \(\frac{25.2 - 1.2}{1.2} \times 100\%\) [also accept \(-95.2\%\) if comparing high relative to low: \(\frac{1.2 - 25.2}{25.2} \times 100\%\)].
Part (d): 2 points maximum
- 1 point for predicting that ZUP1 will be expressed / transcribed / mRNA levels will be high/elevated in the mutant strain in a high-\(\text{Zn}^{2+}\) environment.
- 1 point for providing reasoning that without functional Cbp2, the co-repressor complex fails to assemble/inhibit ZapA, allowing ZapA to bind the promoter and activate transcription.