LEHNINGER PRINCIPLES OF BIOCHEMISTRY - VOL 3. INFORMATION PATHWAYS - 2017

PART III. INFORMATION PATHWAYS

28. REGULATION OF GENE EXPRESSION

Questions and Problems

1. Effect of mRNA and Protein Stability on Regulation.

Suppose E. coli Cells are growing in a medium where Glucose serves as the sole carbon source. Tryptophan is then added to the medium. The cells continue to grow and divide every 30 min. Describe the qualitative changes over time in the level of tryptophan synthase activity in the Cells under the following conditions:

(a) Tryptophan mRNA is stable (degrades slowly over many hours).

(b) Tryptophan mRNA is rapidly degraded, but tryptophan synthase is stable.

(c) Both tryptophan mRNA and tryptophan synthase are rapidly degraded.

2. Negative Regulation.

Describe the possible effects on the Gene Regulation of the lac Operon of the following Mutations: (a) a deletion of a large part of O₁ in the lac operator; (b) a mutation in the lacI gene that inactivates the repressor; (c) an alteration of the region around the -10 position in the promoter.

3. Specificity of DNA Binding by Regulatory Proteins.

A typical bacterial repressor protein binds to its specific DNA target site (the operator) 10⁴ to 10⁶ times more effectively than to a nonspecific DNA sequence. To ensure efficient repression, 10 repressor molecules per Cell are sufficient. Suppose a human cell contains a very similar repressor with the same specificity for its binding site. How many copies of the repressor are required to achieve the same repression efficiency as in a bacterial cell? Hint: The E. coli genome contains about 4.6 million bp; the human haploid genome contains about 3.2 billion bp.

4. Repressor Concentration in E. coli.

The dissociation constant of the repressor-operator complex is very low (about 10^-13 M). An E. coli cell (volume 2 • 10^-12 mL) contains 10 copies of the repressor. Calculate the cellular concentration of the repressor protein. Compare this value with the dissociation constant of the repressor-operator complex. What is The Significance of this result?

5. Catabolite Repression.

E. coli cells are growing in a medium containing lactose but no glucose. Indicate whether each of the following changes or conditions would affect the expression of the lac operon (enhance, diminish, or cause no change). It may be helpful to sketch a model describing each situation.

(a) Addition of a high concentration of glucose.

(b) A mutation that prevents the Lac repressor from dissociating from the operator.

(c) A mutation that completely inactivates β-galactosidase.

(d) A mutation that completely inactivates galactoside permease.

(e) A mutation that prevents CRP from interacting with its binding site near the promoter.

6. METABOLISM/31.html">Transcription Attenuation.

How would Transcription of the E. coli trp operon be affected by the following alterations in the leader region of the tryptophan mRNA?

(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) Deletion of sequence 4.

d) Replacing two Trp codons in the leader peptide gene with His codons.

e) Deletion of the ribosome-binding site in the leader peptide gene.

f) Altering several NUCLEOTIDES in sequence 3 so that it can base-pair with sequence 4, but not with sequence 2.

7. Repressors and Repression.

How might the SOS Response in E. coli be affected by a lexA gene mutation that prevents the autocatalytic Cleavage of the LexA protein?

8. Regulation by Recombination.

What would happen to cells if the Hin recombinase in the Salmonella phase variation system became more active, carrying out recombination (DNA inversion) multiple times per cell generation?

9. Transcription initiation in Eukaryotic cells.

A novel RNA polymerase activity was discovered in a crude extract from an exotic mushroom. This RNA polymerase initiates transcription from a single specialized promoter. Upon purification of the polymerase, its activity progressively decreased, and the purified enzyme was completely inactive unless the crude extract was added back to the reaction mixture. Explain these observations.

10. Functional Domains in Regulatory Proteins.

A biochemist replaced the DNA-binding domain of the Yeast Gal4 protein with the DNA-binding domain of the Lac repressor and found that the engineered protein does not re

gulate the transcription of GAL genes in yeast. Draw a diagram of the functional domains of the wild-type Gal4 protein and the chimeric protein. Why does the chimeric protein fail to regulate GAL gene transcription? What must be changed in the DNA sequence bound by the chimeric protein to enable it to activate GAL gene transcription?

11. Nucleosome Modification during Transcription Activation.

Preparing genomic regions for transcription involves the Acetylation and methylation of specific amino acid residues on Histones within the respective nucleosomes. Once transcription is complete, these modified residues must be reset to their original state. In mammals, the methylation of Arg residues in histones is reversed by the action of peptidylarginine deiminases. However, this reaction yields citrulline rather than unmethylated Arginine. What is the second product of this reaction? Propose a possible mechanism.

12. Mechanisms of Inheritance in Development.

Drosophila eggs with a bcd^-/bcd^- genotype can develop normally, but adult insects with this genotype fail to produce viable offspring. Explain this observation.

Biochemistry on the Internet

13. TATA-Binding Protein and the TATA Box.

To explore the interaction between transcription factors and DNA, access the FirstGlance in Jmol database at http://firstglance.jmol.org and load PDB file ID 1TGH. This file models the interactions between the human TATA-binding protein and a stretch of double-stranded DNA. Once the Structure loads, select the "Spin" option to stop the molecule from rotating. Next, click the "Contacts" link. Under the "Chains" function, click on any part of the protein (chain A, colored blue) to select the corresponding chain. Click "Show Atoms Contacting Target" and, from the appearing list, select the function "Show putatively hydrogen-bonded non-Water" to visualize the Hydrogen Bonds between the protein and the DNA TATA box. Then, click the rightmost button to render the image (Maximum detail: Target & Contacts Balls and Sticks, Colored by Element). You will now be able to zoom in and rotate the structure. Answer the following questions.

a) Which DNA Base Pairs form hydrogen bonds with the protein? Which of these contribute to the specific recognition of the TATA box by the protein? (The Hydrogen bond length between a hydrogen donor and acceptor ranges from 2.5–3.3 Å).

b) Which amino acid residues in the protein interact with these base pairs?

c) What DNA sequence is represented in this model, and which parts of this sequence are recognized by the TATA-binding protein?

d) Identify the regions of hydrophobic interactions in this complex. Are they isolated or numerous? To answer this question, click "Return to contacts" and use the option "Show hydrophobic (apolar Van der Waals) interactions".

Analysis of Experimental Data

14. Engineering a genetic switch in Escherichia coli cells.

Gene Expression regulation is often described in terms of "on/off," implying that a gene is either expressed or not expressed. In reality, ligands play a role in repressing and activating expression, so intermediate levels of regulatory molecules can result in intermediate levels of expression. For example, consider the binding equation for the Lac repressor, operator, and inducer for the lac operon in E. coli (see Fig. 28-7). Although this is a complex cooperative process, to a first approximation it can be described by the following equation (R = repressor, IPTG = inducer, — isopropyl-β-D-thiogalactoside):

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The free repressor R binds to the operator and prevents transcription of the lac operon; the R-IPTG complex does not bind to the operator, and consequently, transcription of the lac operon proceeds.

a) Using equation (5-8), determine the relative expression level of lac operon proteins as a function of the concentra-

tion of the inducer [IPTG]. Use these calculations to determine the range of IPTG concentrations over which the expression level changes from 10% to 90%.

b) Qualitatively estimate The amount of lac operon proteins in E. coli cells before, during, and after The addition of IPTG. You do not need to specify the exact amount of protein at precise time points; simply indicate the trend.

Gardner, Cantor, and Collins (2000) sought to create a "genetic toggle switch"—a gene regulation system with two distinct states, much like an electrical switch. (A) Such a switch has only two positions, on or off; it cannot maintain a dim lighting level. In biochemical terms, this means that the target gene or group of genes (operon) is either fully expressed or not expressed at all, with intermediate expression levels being impossible. (B) Both states are stable; you can flip the switch with your finger, but when you release it, the switch remains in the set position. In biochemical terms, this means that upon exposure to an inducer or other signal, gene or operon expression is turned on or off and remains in that state even after the signal ceases.

c) Explain why the lac operon lacks characteristics A and B.

To construct a "genetic toggle switch," Gardner and colleagues engineered a plasmid consisting of the following elements:

P_lac — operator and promoter region of the E. coli lac operon

O_Rλ — operator and promoter region of phage λ

lacI — gene for the lac repressor, LacI. In the absence of IPTG, this protein completely represses O_Pl_ac; in the presence of IPTG, it allows normal expression from O_Pl_ac.

rep^s — gene for a mutant thermosensitive repressor protein of phage λ, rep^ts. At 37 °C, this protein completely represses O_Rλ; at 42 °C, it allows normal expression from O_Rλ.

GFP — gene for the green fluorescent protein (GFP), a strongly fluorescent marker protein (see Fig. 9-15, Vol. 1)

T — transcription terminator

These elements (see figure) were arranged in such a way that the two promoters mutually repressed each other: O_Pl_ac controlled the expression of rep^ts, and O_Rλ controlled the expression of lacI. The state of the system was evaluated by the level of GFP expression, which was also under the control of O_Pl_ac.

(d) The engineered system could exist in two states: "GFP on" (high expression level) and "GFP off" (low expression level). Describe which proteins are present and which promoters are active in each of the two states.

(e) One would expect Treatment with IPTG to switch the system from one state to the other. From which state to which? Explain your reasoning.

(f) One would also expect raising the Temperature to 42 °C to trigger a transition of the system from one state to the other. From which state to which? Explain your reasoning.

(g) Why did the engineered plasmid have to possess the characteristics A and B described above?

To confirm that the construct indeed possessed these specified characteristics, Gardner and co-workers first showed that once turned on or off, GFP expression (high or low, respectively) remains at that same level over a long period of time (property B). Next, they measured the GFP level at various IPTG concentrations and obtained the following results.

It turned out that the average GFP expression level had some intermediate value at an IPTG concentration equal to X. However, when measuring the GFP expression level in individual cells at [IPTG] = X, it was found that the GFP level was either low or high, and not a single cell showed an intermediate level of expression of this protein.

c) Explain how this observation confirms the fact that the system exhibits property A? Why is a high level of marker protein expression observed in some cells and a low level in others at [IPTG] = X?