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Ch. 16 - Regulation of Gene Expression in Bacteria
Klug - Concepts of Genetics 12th Edition
Klug12th EditionConcepts of Genetics ISBN: 9780135564776Not the one you use?Change textbook
All textbooksKlug 12th EditionCh. 16 - Regulation of Gene Expression in BacteriaProblem 22b
Chapter 16, Problem 22b

The SOS repair genes in E. coli are negatively regulated by the lexA gene product, called the LexA repressor. When a cell's DNA sustains extensive damage, the LexA repressor is inactivated by the recA gene product (RecA), and transcription of the SOS genes is increased dramatically. One of the SOS genes is the uvrA gene. You are a student studying the function of the uvrA gene product in DNA repair. You isolate a mutant strain that shows constitutive expression of the UvrA protein. Naming this mutant strain uvrAC, you construct the diagram shown above in the right-hand column showing the lexA and uvrA operons:
Outline a series of genetic experiments that would use partial diploid strains to determine which of the two possible mutations you have isolated.
Diagram of lexA and uvrA operons showing promoter, operator, and gene regions for analyzing uvrAC mutant expression.

Verified step by step guidance
1
Understand the problem: The uvrAᶜ mutant strain shows constitutive expression of the UvrA protein, meaning the gene is always active regardless of DNA damage. The goal is to determine whether the mutation is in the uvrA gene itself (e.g., in its promoter or operator) or in the lexA gene (which encodes the LexA repressor). Partial diploid strains can help distinguish between these possibilities.
Construct a partial diploid strain: Introduce a wild-type copy of the uvrA gene into the uvrAᶜ mutant strain. This can be done by introducing a plasmid carrying the wild-type uvrA gene. Observe whether the constitutive expression of UvrA persists. If the mutation is in the uvrA gene itself (e.g., in its operator or promoter), the wild-type copy will not suppress the constitutive expression. If the mutation is in the lexA gene, the wild-type lexA gene will restore normal regulation.
Construct another partial diploid strain: Introduce a wild-type copy of the lexA gene into the uvrAᶜ mutant strain. This can be done by introducing a plasmid carrying the wild-type lexA gene. Observe whether the constitutive expression of UvrA is suppressed. If the mutation is in the lexA gene, the wild-type copy will restore normal regulation. If the mutation is in the uvrA gene, the constitutive expression will persist.
Analyze the results: Compare the expression of UvrA in the two partial diploid strains. If the wild-type uvrA gene does not suppress constitutive expression but the wild-type lexA gene does, the mutation is in the lexA gene. If neither wild-type gene suppresses constitutive expression, the mutation is likely in the uvrA gene itself.
Confirm the findings: To further confirm the location of the mutation, sequence the uvrA and lexA genes in the mutant strain. Look for mutations in the promoter or operator region of uvrA or in the coding region of lexA. This will provide definitive evidence for the location of the mutation.

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Key Concepts

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

SOS Response

The SOS response is a cellular mechanism in bacteria, particularly E. coli, that is activated in response to extensive DNA damage. This response involves the expression of a set of genes, including the uvrA gene, which are crucial for DNA repair. The process is regulated by the LexA repressor, which inhibits the transcription of these genes under normal conditions. When DNA damage occurs, the RecA protein facilitates the inactivation of LexA, leading to the upregulation of SOS genes.
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LexA Repressor

The LexA repressor is a protein that negatively regulates the SOS response in E. coli by binding to the operator regions of SOS genes, preventing their transcription. Under normal circumstances, LexA maintains low levels of SOS gene expression. However, when DNA damage is detected, RecA binds to single-stranded DNA and promotes the autocleavage of LexA, thereby relieving repression and allowing for the expression of genes necessary for DNA repair, including uvrA.
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Partial Diploids and Genetic Experiments

Partial diploids are bacterial strains that contain two copies of certain genes, typically achieved through the introduction of a plasmid or a second chromosome segment. This genetic setup is useful for determining the dominance or recessiveness of mutations. In the context of the uvrAᶜ mutant strain, constructing partial diploids can help elucidate whether the constitutive expression of UvrA is due to a mutation in the uvrA gene itself or in the regulatory lexA gene, allowing for a clearer understanding of the genetic interactions involved.
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Related Practice
Textbook Question

A bacterial operon is responsible for the production of the biosynthetic enzymes needed to make the hypothetical amino acid tisophane (tis). The operon is regulated by a separate gene, R. The deletion of R causes the loss of enzyme synthesis. In the wild-type condition, when tis is present, no enzymes are made; in the absence of tis, the enzymes are made. Mutations in the operator gene (O⁻) result in repression regardless of the presence of tis. Is the operon under positive or negative control? Propose a model for:

(a) Repression of the genes in the presence of tis in wild-type cells

(b) The mutations.

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Textbook Question

A marine bacterium is isolated and shown to contain an inducible operon whose genetic products metabolize oil when it is encountered in the environment. Investigation demonstrates that the operon is under positive control and that there is a reg gene whose product interacts with an operator region (o) to regulate the structural genes, designated sg. In an attempt to understand how the operon functions, a constitutive mutant strain and several partial diploid strains were isolated and tested with the results shown in the following table. Host Chromosome F' Factor Phenotype Wild type None Inducible Wild type reg gene from mutant strain Inducible Wild type Operon from mutant strain Constitutive Mutant strain reg gene from wild type Constitutive Draw all possible conclusions about the mutation as well as the nature of regulation of the operon. Is the constitutive mutation in the trans-acting reg element or in the cis-acting o operator element?

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Textbook Question

The SOS repair genes in E. coli are negatively regulated by the lexA gene product, called the LexA repressor. When a cell's DNA sustains extensive damage, the LexA repressor is inactivated by the recA gene product (RecA), and transcription of the SOS genes is increased dramatically. One of the SOS genes is the uvrA gene. You are a student studying the function of the UvrA gene product in DNA repair. You isolate a mutant strain that shows constitutive expression of the UvrA protein. Naming this mutant strain uvrAᶜ, you construct the diagram shown above in the right-hand column showing the lexA and uvrA operons:

Describe two different mutations that would result in a uvrA constitutive phenotype. Indicate the actual genotypes involved.

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Textbook Question

A fellow student considers the issues in Problem 22 and argues that there is a more straightforward, nongenetic experiment that could differentiate between the two types of mutations. The experiment requires no fancy genetics and would allow you to easily assay the products of the other SOS genes. Propose such an experiment.

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Textbook Question

The following figure depicts numerous critical regions of the leader sequence of mRNA that play important roles during the process of attenuation in the trp operon. A closer view of the leader sequence, which begins at about position 30 downstream from the 5' end, is shown below, running along both columns. Within this molecule are the sequences that cause the formation of the alternative hairpins. It also contains the successive triplets that encode tryptophan, where stalling during translation occurs.

Take a large piece of paper (such as manila wrapping paper) and, along with several other students from your genetics class, work through the base sequence to identify the trp codons and the parts of the molecule representing the base-pairing regions that form the terminator and antiterminator hairpins shown in the following figure.

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