Chapter 26

The active form of lactose repressor binds to the operator with a dissociation constant of \(10^{-13} \mathrm{M}\) for the reaction \(\mathrm{R}+\mathrm{O} \rightleftharpoons \mathrm{RO}\). About 10 molecules per \(E\). coli cell suffice to keep the operon turned off in the absence of inducer. (a) If the average \(E\). coli cell has an intracellular volume of \(0.3 \times 10^{-12} \mathrm{~mL}\), calculate the approximate intracellular concentration of repressor. (b) If the average cell contains two copies of the lac operon, calculate the approximate intracellular concentration of operators. (c) Calculate the average intracellular concentration of free operators under these conditions. (d) Explain how a cell with a haploid chromosome could contain an average of two copies of the lac operon.

Is attenuation likely to be involved in eukaryotic gene regulation? Briefly explain your answer.

What are the major differences between an operon and a regulon?

For some time, it was not clear whether lac repressor inhibits lac operon transcription by inhibiting the binding of RNA polymerase to its promoter or by allowing transcription initiation but blocking elongation past the site of bound repressor. How might you distinguish between these possibilities?

A lac operon containing one mutation was cloned into a plasmid, which was introduced by transformation into a bacterium containing a wild-type lac operon. The three genes of the chromosomal operon were rendered noninducible in the presence of the plasmid. (a) What kind of mutation in the plasmid operon could have this effect? (b) Suppose the result of transformation was to cause the three plasmid lac genes to be expressed constitutively, at a high level. What type of plasmid gene mutation could have this result?

Repressors are inactivated either by interaction with a small-molecule inducer or by proteolytic cleavage. Why is it advantageous for a repressor like the lac repressor to be inactivated by binding to allolactose rather than by proteolytic cleavage?

Partial diploid forms of \(E\). coli were created, each of which contained a complete lactose operon at its normal chromosomal site and the regulatory sequences only (lacl, \(P\), lac \(O\) ) on a plasmid. Predict the effect of each mutation on the activity of \(\beta\)-galactosidase before and after the addition of inducer. Use \(-,+\), or \(++\) to indicate approximate activity levels. Briefly explain the basis for each of your predictions. \begin{tabular}{l} Mutation \\ (a) No mutations in either chromosomal or plasmid genes \\ (b) A mutation in the plasmid operator, which abolishes \\ its binding to repressor \\ (c) A mutation in the chromosomal promoter, which reduces \\ (d) A lacl gene mutation in the chromosome, which abol- \\ ishes binding of the lacl gene product to inducer \\ (e) A lacl gene mutation in the plasmid, which abolishes \\ (f) A chromosomal lac O mutation, which abolishes its \\ (g) A mutation in the gene for CRP, which abolishes its \\ \hline \end{tabular}

What type of mutation of the lac repressor might be both constitutive and trans-dominant?

It has been proposed that thiogalactoside transacetylase (LacA in the lactose operon) plays a role in detoxification-ridding the cell of potentially toxic \(\beta\)-galactosides by acetylating them to inhibit their reuptake after their diffusion out of the cell. How might you test this proposal?

In eukaryotic transcription, what is the function of a histone chaperone? Of a chromatin remodeling complex? Of Mediator?

Why does histone deacetylase action tend to repress transcription?

Explain how a gene knockdown reagent, such as RNAi or a morpholino, could interfere with expression of a gene without affecting the rate of transcription of the target gene.

Briefly explain how heme regulates the expression of globin genes in cells that synthesize hemoglobin.

What condition must be fulfilled in order for histone modification to be considered an epigenetic phenomenon? Does DNA methylation meet that criterion? Explain.

When binding of sequence-specific regulatory proteins such as lac repressor or CRP to its respective DNA binding site was studied in vitro, the rates of association were much higher than predicted by collision theory. Propose an explanation for this observation and a simple experimental test of your hypothesis.

Cancer is thought to result in part from an increase in the spontaneous mutation rate in precancerous cells. At the same time, the DNA methylation pattern changes during oncogenic transformation. Identify a process by which DNA methylation might lead to increased mutagenesis.