Problem 1
Effect of mRNA and Protein Stability on Regulation \(E\). coli cells are growing in a medium with glucose as the sole carbon source. After the sudden addition of tryptophan, the cells continue to grow and divide every \(30 \mathrm{~min}\). Describe (qualitatively) how the amount of tryptophan synthase activity in the cells changes with time under each condition: a. The trp mRNA is stable (degrades slowly over many hours). b. The \(\operatorname{trp}\) mRNA degrades rapidly, but tryptophan synthase is stable. c. The \(\operatorname{trp}\) mRNA and tryptophan synthase both degrade rapidly.
Problem 3
Negative Regulation Describe the probable effects on gene expression in the lac operon of each mutation: a. Mutation in the lac operator that deletes most of \(\mathrm{O}_{1}\) b. Mutation in the lacI gene that eliminates binding of repressor to operator c. Mutation in the promoter near position \(-10\) that increases its similarity to the \(E\). coli consensus sequence d. Mutation in the lacI gene that eliminates binding of repressor to lactose e. Mutation in the promoter near position \(-10\) that decreases its similarity to the \(E\). coli consensus sequence
Problem 4
Specific DNA Binding by Regulatory Proteins A typical bacterial repressor protein discriminates between its specific DNA-binding site (operator) and nonspecific DNA by a factor of \(10^{4}\) to \(10^{6}\). About 10 molecules of repressor per cell are sufficient to ensure a high level of repression. Assume that a very similar repressor existed in a human cell, with a similar specificity for its binding site. How many copies of the repressor would a human cell require to elicit a level of repression similar to that in the bacterial cell? (Hint: The \(E\). coli genome contains about \(4.6\) million bp; the human haploid genome has about \(3.2\) billion bp.)
Problem 5
Repressor Concentration in \(\boldsymbol{E}\). coli The dissociation constant for a particular repressor-operator complex is very low, about \(10^{-13}\) M. An \(E\). coli cell (volume \(2 \times 10^{-12} \mathrm{~mL}\) ) contains 10 copies of the repressor. Calculate the cellular concentration of the repressor protein. How does this value compare with the dissociation constant of the repressoroperator complex? What is the significance of this answer?
Problem 6
Catabolite Repression \(E\). coli cells are growing in a medium that contains lactose but no glucose. Indicate whether each of the following changes or conditions would increase, decrease, or not change the expression of the lac operon. It may be helpful to draw a model depicting what is happening in each situation. a. Addition of a high concentration of glucose b. A mutation that prevents dissociation of the Lac repressor from the operator c. A mutation that completely inactivates \(\beta\) galactosidase d. A mutation that completely inactivates galactoside permease e. A mutation that prevents binding of CRP to its binding site near the lac promoter
Problem 7
Transcription Attenuation How would each manipulation of the leader region of the \(\operatorname{trp}\) mRNA affect transcription of the \(E\). coli trp operon? a. Increasing the distance (number of bases) between the leader peptide gene and sequence 2 b. Increasing the distance between sequences 2 and 3 c. Removing sequence 4 d. Changing the two Trp codons in the leader peptide gene to His codons e. Eliminating the ribosome-binding site for the gene that encodes the leader peptide f. Changing several nucleotides in sequence 3 so that it can base-pair with sequence 4 but not with sequence 2
Problem 8
Repressors and Repression How would a mutation in the lexA gene that prevents autocatalytic cleavage of the LexA protein affect the SOS response in \(E\). coli?
Problem 9
Regulation by Recombination In the phase variation system of Salmonella, what would happen to the cell if the Hin recombinase became more active and promoted recombination (DNA inversion) several times in each cell generation?
Problem 10
Initiation of Transcription in Eukaryotes A biochemist discovers a new RNA polymerase activity in crude extracts of cells derived from an exotic fungus. The RNA polymerase initiates transcription only from a single, highly specialized promoter. As the biochemist purifies the polymerase, its activity declines, and the purified enzyme is completely inactive unless he adds crude extract to the reaction mixture. Suggest an explanation for these observations.
Problem 12
Nucleosome Modification during Transcriptional Activation To prepare genomic regions for transcription, cells acetylate and methylate certain histones in the resident nucleosomes at specific locations. Once transcription is no longer needed, cells need to reverse these modifications. In mammals, peptidylarginine deiminases (PADIs) reverse the methylation of Arg residues in histones. The reaction promoted by these enzymes does not yield unmethylated arginine. Instead, it produces citrulline residues in the histone. What is the other product of the reaction? Suggest a mechanism for this reaction.
