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Chapter 16: Problem 8

Describe the experimental rationale that allowed the lac repressor to be isolated.

Short Answer

Expert verified
Answer: The key steps in isolating and characterizing the lac repressor protein in bacteria include understanding the lac operon system, identifying bacterial strains with lac repressor mutations, performing genetic crosses and analyzing patterns of inheritance, isolating mutant lac repressor proteins, studying their biochemical properties, and characterizing the lac repressor gene sequence.

Step by step solution

01

Understanding the lac operon system

The lac repressor is a protein that controls the lac operon in bacteria, a set of genes responsible for transporting and metabolizing lactose. When lactose is present, it binds to the lac repressor, causing a conformational change and allowing gene expression. When lactose is absent, the lac repressor binds to the operator sequence of the lac operon, preventing RNA polymerase from transcribing the genes.
02

The experimental rationale

To isolate the lac repressor, researchers used a genetic approach to identify the repressor. They first identified bacterial strains with a mutation that led to the continuous expression of the lac operon, even in the absence of lactose. This constitutive expression indicated that the repressor was non-functional, pointing to the presence of a mutation in the lac repressor gene itself.
03

Identification of lac repressor mutations

Scientists isolated these mutant bacterial strains and performed genetic crosses with other strains carrying different mutations affecting the lac operon. By analyzing the patterns of inheritance, researchers could differentiate between mutations affecting the repressor and those affecting other elements of the lac operon, such as the operator sequence or the structural genes.
04

Isolation of mutant lac repressor proteins

Since the lac repressor is a protein, the next step was to identify and characterize the corresponding mutated proteins. Researchers used techniques such as gel electrophoresis and affinity chromatography to isolate the proteins, comparing them to wild-type repressor proteins to determine any structural differences caused by the mutations.
05

Studying the biochemical properties of the lac repressor

With the mutated lac repressor proteins isolated, researchers could study their biochemical properties. This included determining their binding specificity, measuring their affinity for the operator sequence, and analyzing any changes in their three-dimensional structure. These experiments would provide further evidence that the isolated proteins were indeed the lac repressor and would help characterize the functional properties of the protein, leading to a better understanding of its role in the lac operon system.
06

Characterization of the lac repressor gene sequence

To identify the gene sequence encoding the lac repressor protein, scientists isolated the DNA region corresponding to the mutant strains and compared it to the DNA region from wild-type strains. By identifying the specific mutations present in the repressor gene, they could further correlate these genetic changes with the altered protein properties observed in the mutant strains. This provided a complete picture of the relationship between the repressor protein and its gene.

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Most popular questions from this chapter

In this chapter, we focused on the regulation of gene expression in prokaryotes. Along the way, we found many opportunities to consider the methods and reasoning by which much of this information was acquired. From the explanations given in the chapter, what answers would you propose to the following fundamental questions? (a) How do we know that bacteria regulate the expression of certain genes in response to the environment? (b) What evidence established that lactose serves as the inducer of a gene whose product is related to lactose metabolism? (c) What led researchers to conclude that a repressor molecule regulates the lac operon? (d) How do we know that the lac repressor is a protein? (e) How do we know that the trp operon is a repressible con- trol system, in contrast to the lac operon, which is an inducible control system?

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, \(R,\) deletion of which 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 and (b) the mutations.

Predict the effect on the inducibility of the \(\operatorname{lac}\) operon of a mutation that disrupts the function of (a) the crp gene, which encodes the CAP protein, and (b) the CAP-binding site within the promoter.

Describe the role of attenuation in the regulation of tryptophan biosynthesis.

The SOS repair genes in \(E\). coli (discussed in Chapter 15 ) are negatively regulated by the lexA gene product, called the LexA (a) Describe two different mutations that would result in a \(u v r A\) constitutive phenotype. Indicate the actual genotypes involved. (b) Outline a series of genetic experiments that would use partial diploid strains to determine which of the two possible mutations you have isolated.
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