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Ch. 17+18 - Transcriptional Regulation in Eukaryotes
All textbooksKlug 12th EditionCh. 17+18 - Transcriptional Regulation in EukaryotesProblem 27
Chapter 17, Problem 27

During an examination of the genomic sequences surrounding the human β-globin gene, you discover a region of DNA that bears sequence resemblance to the glucocorticoid response element (GRE) of the human metallothionein IIA (hMTIIA) gene. Describe experiments that you would design to test (1) whether this sequence was necessary for accurate β-globin gene expression and (2) whether this sequence acted in the same way as the hMTIIA gene's GRE.

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Design a deletion analysis experiment to test necessity: Create a series of constructs where the suspected GRE-like sequence is deleted from the β-globin gene promoter region.
Transfect these constructs into a suitable cell line that expresses the β-globin gene and measure the expression levels of the β-globin gene using quantitative PCR or reporter assays.
Compare the expression levels of the β-globin gene in constructs with and without the GRE-like sequence to determine if the sequence is necessary for expression.
To test if the sequence acts like the hMTIIA gene's GRE, perform a DNA-binding assay such as Electrophoretic Mobility Shift Assay (EMSA) to see if glucocorticoid receptor proteins bind to the sequence.
Conduct a reporter assay by cloning the GRE-like sequence upstream of a minimal promoter driving a reporter gene, and treat cells with glucocorticoids to see if the sequence enhances expression, similar to the hMTIIA GRE.

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Key Concepts

Here are the essential concepts you must grasp in order to answer the question correctly.

Gene Expression Regulation

Gene expression regulation involves mechanisms that control the timing and amount of gene product (RNA or protein) produced. This can include transcription factors binding to specific DNA sequences, such as enhancers or response elements, which can either promote or inhibit transcription. Understanding how these regulatory elements function is crucial for determining their necessity in gene expression.
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Transcription Factor Binding Assays

Transcription factor binding assays, such as electrophoretic mobility shift assays (EMSAs) or chromatin immunoprecipitation (ChIP), are used to study the interaction between transcription factors and specific DNA sequences. These experiments can help identify whether the GRE-like sequence interacts with transcription factors that regulate β-globin expression, providing insight into its functional role.
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Reporter Gene Assays

Reporter gene assays are experimental techniques used to measure the activity of a specific promoter or regulatory element by linking it to a reporter gene, such as luciferase or GFP. By constructing plasmids with the β-globin gene and the GRE-like sequence, researchers can assess whether this sequence influences β-globin expression levels, allowing for comparisons with the hMTIIA GRE's effects.
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Related Practice
Textbook Question

Regulation of the lac operon in E. coli (see Chapter 16) and regulation of the GAL system in yeast are analogous in that they both serve to adapt cells to growth on different carbon sources. However, the transcriptional changes are accomplished very differently. Consider the conceptual similarities and differences as you address the following.

Compare and contrast how these two systems are negatively regulated such that they are downregulated in the presence of glucose.

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Textbook Question

Incorrectly spliced RNAs often lead to human pathologies. Scientists have examined cancer cells for splice-specific changes and found that many of the changes disrupt tumor-suppressor gene function [Xu and Lee (2003). Nucl. Acids Res. 31:5635–5643]. In general, what would be the effects of splicing changes on these RNAs and the function of tumor-suppressor gene function? How might loss of splicing specificity be associated with cancer?

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Textbook Question

Mutations in the low-density lipoprotein receptor (LDLR) gene are a primary cause of familial hypercholesterolemia. One such mutation is a SNP in exon 12 of the LDLR. In premenopausal women, but not in men or postmenopausal women, this SNP leads to skipping of exon 12 and production of a truncated nonfunctional protein. It is hypothesized that this SNP compromises a splice enhancer [Zhu et al. (2007). Hum Mol Genet. 16:1765–1772]. What are some possible ways in which this SNP can lead to this defect, but only in premenopausal women?

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Textbook Question

RNA helicases are a class of proteins that bind mRNAs and influence their secondary structures and interactions with other proteins. RNA helicases have been implicated in many steps of RNA regulation such as splicing, decay, and translation. Why might these enzymes be so ubiquitously required for RNA regulation?

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Textbook Question

Marine stickleback fish have pelvic fins with long spines that provide protection from larger predatory fish. Some stickleback fish were trapped in lakes and have adapted to life in a different environment. Many lake populations of stickleback fish lack pelvic fins. Shapiro et al. (2004) (Nature 428:717.723) mapped the mutation associated with the loss of pelvic fins to the Pitx1 locus, a gene expressed in pelvic fins, the pituitary gland, and the jaw. However, the coding sequence of the Pitx1 gene is identical in marine and lake stickleback [Chan et al. (2010). Science 327:5963,302–305]. Moreover, when the Pitx1 coding region is deleted, the fish die with defects in the pituitary gland and the jaw, and they lack pelvic fins. Explain how a mutation near, but outside of, the coding region of Pitx1 may cause a loss of pelvic fins without pleiotropic effects on the pituitary gland and jaw.

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Textbook Question
List three different types of posttranslational modifications that may happen to a protein and the significance of each in the context of protein function.
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