In this chapter, we focused on the regulation of gene expression in bacteria. 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? How do we know that bacteria regulate the expression of certain genes in response to the environment?

What evidence established that lactose serves as the inducer of a gene whose product is related to lactose metabolism?

What led researchers to conclude that a repressor molecule regulates the lac operon?

How do we know that the lac repressor is a protein?

How do we know that the trp operon is a repressible control system, in contrast to the lac operon, which is an inducible control system?

Write a brief essay that discusses why you think regulatory systems evolved in bacteria (i.e., what advantages do regulatory systems provide to these organisms?), and, in the context of regulation, discuss why genes related to common functions are found together in operons.

Contrast positive versus negative control of gene expression.

Contrast the role of the repressor in an inducible system and in a repressible system.

For the lac genotypes shown in the following table, predict whether the structural genes (Z) are constitutive, permanently repressed, or inducible in the presence of lactose. Genotype Constitutive Repressed Inducible I⁺O⁺Z⁺ x I⁻O⁺Z⁺ I⁻OᶜZ⁺ I⁻OᶜZ⁺/F'O⁺ I⁺OᶜZ⁺/F'O⁺ IˢO⁺Z⁺ IˢO⁺Z⁺/F'I⁺

For the genotypes and conditions (lactose present or absent) shown in the following table, predict whether functional enzymes, nonfunctional enzymes, or no enzymes are made.

<IMAGE>

The locations of numerous lacI⁻ and lacIˢ mutations have been determined within the DNA sequence of the lacI gene. Among these, lacI⁻ mutations were found to occur in the 5′-upstream region of the gene, while lacIˢ mutations were found to occur farther downstream in the gene. Are the locations of the two types of mutations within the gene consistent with what is known about the function of the repressor that is the product of the lacI gene?

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

What properties demonstrate that the lac repressor is a protein? Describe the evidence that it indeed serves as a repressor within the operon system.

Predict the effect on the inducibility of the lac operon of a mutation that disrupts the function of:

(a) The CRP gene, which encodes the CAP protein

(b) The CAP-binding site within the promoter.

Erythritol, a natural sugar abundant in fruits and fermenting foods, is about 65 percent as sweet as table sugar and has about 95 percent fewer calories. It is 'tooth friendly' and generally devoid of negative side effects as a human consumable product. Pathogenic Brucella strains that catabolize erythritol contain four closely spaced genes, all involved in erythritol metabolism. One of the four genes (eryD) encodes a product that represses the expression of the other three genes. Erythritol catabolism is stimulated by erythritol. Present a simple regulatory model to account for the regulation of erythritol catabolism in Brucella. Does this system appear to be under inducible or repressible control?

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

What is the major difference between the mechanism involved in attenuation and riboswitches and the mechanism involved in the regulation of the lactose operon?

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.

Bacterial sRNAs can bind to mRNAs through complementary binding to regulate gene expression. What determines whether the sRNA/mRNA binding will promote or repress mRNA translation?

Why is the CRISPR-Cas system of bacteria considered an adaptive immunity rather than an innate immunity?

How does the molecular mechanism of the CRISPR-Cas system use a viral DNA sequence against that same virus?

In the publication that provided the first evidence of CRISPR-Cas as an adaptive immune system [Barrangou, R., et al. (2007). Science. 315:1709–1712], the authors state that CRISPR-Cas “provides a historical perspective of phage exposure, as well as a predictive tool for phage sensitivity.” Explain how this is true using what you know about the CRISPR locus.