Compare the structure of IPTG shown in Figure 11-7 with the structure of galactose shown in Figure 11-5. Why is IPTG bound by the Lac repressor but not broken down by β-galactosidase?

Looking at Figure 11-9, why were partial diploids essential for establishing the trans-acting nature of the Lac repressor? Could one distinguish cis-acting from trans-acting genes in haploids?

Why do promoter mutations cluster at positions −10 and −35 as shown in Figure 11-11? Which protein-DNA interaction is disrupted by these mutations?

Looking at Figure 11-16, note the large overlap between the operator and the region of the lac operon that is transcribed. Which protein binds specifically to this overlapping sequence, and what effect does it have on transcription?

Examining Figure 11-21, what effect do you predict trpA mutations will have on tryptophan levels?

Examining Figure 11-21, what effect do you predict trpA mutations will have on trp mRNA expression?

Which of the following molecules is an inducer of the lac operon:

Explain why I⁻ alleles in the lac system are normally recessive to I⁺ alleles and why I⁺ alleles are recessive to I^S alleles.

What do we mean when we say that O^C mutations in the lac system are cis-acting?

The symbols a, b, and c in the table below represent the E. coli lac system genes for the repressor (I), the operator (O) region, and the β-galactosidase (Z), although not necessarily in that order. Furthermore, the order in which the symbols are written in the genotypes is not necessarily the actual sequence in the lac operon.

Activity (+) or inactivity (−) of Z gene

The map of the lac operon is

POZY

The promoter (P) region is the start site of transcription through the binding of the RNA polymerase molecule before actual mRNA production. Mutationally altered promoters (P⁻) apparently cannot bind the RNA polymerase molecule. Certain predictions can be made about the effect of P⁻ mutations. Use your predictions and your knowledge of the lactose system to complete the following table. Insert a “+” where an enzyme is produced and a “−” where no enzyme is produced. The first one has been done as an example.

Explain the fundamental differences between negative control and positive control of transcription in prokaryotes. Cite two examples of each control mechanism.

Mutants that are lacY⁻ retain the capacity to synthesize β-galactosidase. However, even though the lacI gene is still intact, β-galactosidase can no longer be induced by adding lactose to the medium. Explain.

What are the analogies between the mechanisms controlling the lac operon and those controlling phage λ genetic switches?

Compare the arrangement of cis-acting sites in the control regions of the lac operon and phage λ.

Which regulatory protein induces the lytic phase genes of the phage λ life cycle?

Predict the effect of a mutation that eliminates the DNA-binding activity of the σ^E protein on spore formation in Bacillus subtilis.

An interesting mutation in lacI results in repressors with 110-fold increased binding to both operator and nonoperator DNA. These repressors display a “reverse” induction curve, allowing β-galactosidase synthesis in the absence of an inducer (IPTG) but partly repressing β-galactosidase expression in the presence of IPTG. How can you explain this? (Note that, when IPTG binds a repressor, it does not completely destroy operator affinity, but rather it reduces affinity 110-fold. Additionally, as cells divide and new operators are generated by the synthesis of daughter strands, the repressor must find the new operators by searching along the DNA, rapidly binding to nonoperator sequences and dissociating from them.)

Certain lacI mutations eliminate operator binding by the Lac repressor but do not affect the aggregation of subunits to make a tetramer, the active form of the repressor. These mutations are partly dominant over wild type. Can you explain the partly dominant I⁻ phenotype of the I⁻/I⁺ heterodiploids?

You are examining the regulation of the lactose operon in the bacterium Escherichia coli. You isolate seven new independent mutant strains that lack the products of all three structural genes. You suspect that some of these mutations are lacI^S mutations and that other mutations are alterations that prevent the binding of RNA polymerase to the promoter region. Using whatever haploid and partial diploid genotypes that you think are necessary, describe a set of genotypes that will permit you to distinguish between the lacI and lacP classes of uninducible mutations.

You are studying the properties of a new kind of regulatory mutation of the lactose operon. This mutation, called S, leads to the complete repression of the lacZ, lacY, and lacA genes, regardless of whether lactose is present. The results of studies of this mutation in partial diploids demonstrate that this mutation is completely dominant over wild type. When you treat bacteria of the S mutant strain with a mutagen and select for mutant bacteria that can express the enzymes encoded by lacZ, lacY, and lacA genes in the presence of lactose, some of the mutations map to the lac operator region and others to the lac repressor gene. On the basis of your knowledge of the lactose operon, provide a molecular genetic explanation for all these properties of the S mutation. Include an explanation of the constitutive nature of the “reverse mutations.”

The trp operon in E. coli encodes enzymes essential for the biosynthesis of tryptophan. The general mechanism for controlling the trp operon is similar to that observed with the lac operon: when the repressor binds to the operator, transcription is prevented; when the repressor does not bind to (as in, to the operator), like phrase just before the operator, transcription proceeds. The regulation of the trp operon differs from the regulation of the lac operon in the following way: the enzymes encoded by the trp operon are not synthesized when tryptophan is present but rather when it is absent. In the trp operon, the repressor has two binding sites: one for DNA and the other for the effector molecule, tryptophan. The trp repressor must first bind to a molecule of tryptophan before it can bind effectively to the trp operator.

The activity of the enzyme β-galactosidase produced by wild-type cells grown in media supplemented with different carbon sources is measured. In relative units, the following levels of activity are found:

Predict the relative levels of β-galactosidase activity in cells grown under similar conditions when the cells are lacI⁻, lacI^S, lacO⁺, and crp⁻.

A bacteriophage λ is found that is able to lysogenize its E. coli host at 30°C but not at 42°C. What genes may be mutant in this phage?

What would happen to the ability of bacteriophage λ to lyse a host cell if it acquired a mutation in the O_R binding site for the Cro protein? Why?

Contrast the effects of mutations in genes encoding sporulation-specific σ factors with mutations in the −35 and −10 regions of the promoters of genes in their regulons.