Biologia molekularna, Lista 6

BIOLOGIA MOLEKULARNA - Seminarium

LISTA 6

Kontrola ekspresji genów u Prokaryota

1. Which of the following statements about β-galactosidase in E. coli are correct?

(a) It is present in varying concentrations, depending on the carbon source used for growth.

(b) It is a product of a unit of gene expression called an operon.

(d) It forms the β-1,6-linked disaccharide allolactose.

(e) It is activated allosterically by the nonmetabolizable compound isopropylthiogalactoside (IPTG).

(f) Its levels rise coordinately with those of galactoside permease and thiogalactoside transacetylase.

2. Match each feature or function in the right column with the appropriate DNA sequence element of the lac operon in the left column.

(a) i

(1) contains a specific binding sequence for the lac repressor

(b) p

(2) encodes a galactoside permease

(3) contains a binding sequence for the cAMP—CAP complex

(d) z

(4) encodes a protein that interferes with the activation of RNA polymerase

(e) y and a

(5) encodes a protein that binds allolactose

(f) CAP binding site

(6) contains a specific binding sequence for RNA polymerase

(7) encodes β-galactosidase

(8) is a regulatory gene

(9) is the lac promoter

(10) encodes thiogalactoside transacetylase

(11) is the lac operator

(12) encodes the lac repressor

3. When E. coli is added to a culture containing both lactose and glucose, which of the sugars is metabolized preferentially? What is the mechanism underlying this selectivity?

4. What happens after the first-used sugar is depleted during the experiment described in the previous question?

5. Which of the following statements about the cAMP—CAP complex are correct?

(a) It protects the –87 to –49 sequence of the lac operon from nuclease digestion.

(b) It protects the –48 to +5 sequence of the lac operon from nuclease digestion.

(d) It affects RNA polymerase activity in a number of operons.

(e) Upon binding to the lac operon, it contacts RNA polymerase.

6. Some of the known constitutive mutations of the lactose operon occur in the operator sequence rather than the regulator gene.

(a) Would you expect such an oc mutant to be dominant or recessive to its wild-type o+ allele? Explain.

(b) Is a constitutive mutation in an operator cis-acting or trans-acting in its effects? Explain.

(c) Design an experiment involving the genes i+, oc, o+, and z+ that would confirm your answer to part (b). Assume that it is possible to detect whether enzymes are produced in diploid (++) or haploid (+) amounts.

BIOLOGIA MOLEKULARNA – Lista 6 – Kontrola ekspresji genów u Prokaryota.

7. Regulation of the trp operon involves which of the following?

(a) Controlling the amount of polycistronic mRNA formed at the level of transcription initiation.

(b) Controlling the amount of polycistronic mRNA at the level of transcription termination.

(d) The sequential and coordinate production of five enzymes of tryptophan metabolism from five different mRNAs produced in equal concentrations.

(e) The production of transcripts of different sizes, depending on the level of tryptophan in the cell.

8. Which of the following statements concerning the trp operon leader RNA, which has 162 nucleotides preceding the initiation codon of the first structural gene of the operon, are correct?

(a) A deletion mutation in the DNA encoding the 3’ region of the leader RNA gives rise to increased levels of the biosynthetic enzymes forming Trp.

(b) A short open reading frame, containing Trp codons among others, exists within the leader RNA.

(d) The leader RNA may form two alternative and mutually exclusive secondary structures.

(e) The structure of the leader RNA in vivo depends on the position of the ribosomes translating it.

9. In order to prove that regulation by attenuation occurs in vivo, Charles Yanofsky and others studied tryptophan synthesis regulation in a series of E. coli mutants. For each mutant described below predict the expression of tryptophan synthesis genes in the presence or absence of tryptophan.

Mutant A: mutations with decreased, but detectable, tryptophanyl-tRNA synthetase activity

Mutant B: mutations in AUG or Shine-Dalgarno sequence of leader peptide sequence

Mutant C: same as mutant B but with the leader peptide fully expressed on a plasmid

Mutant D: a G·C to A·T transversion mutation that weakens the segment 2/segment 3 interaction without affecting the segment 1/segment 2 interaction

Mutant E: mutations replacing the two Trp codons with Leu codons

Mutant F: mutant E with a mutation in the Leu-tRNA synthetase gene

Mutant G: mutant E that constitutively synthesizes leucine

10. Which of the following statements about bacteriophage λ during lytic phase are correct?

(a) It synthesizes three different classes of mRNA, which are designated by the times after infection they appear.

(b) It carries out a programmed synthesis of the proteins necessary to replicate its genome and produce its structural components.

(c) It forms N and Q gene products, which act as positive regulatory proteins, leading to the sequential production of λ-encoded proteins.

(d) It initially produces two proteins; one acts as an inhibitor of λ repressor synthesis, and the other acts as a transcription termination factor.

11. Suppose that the relative affinities of the three O regulatory sequences and the three

regulatory sequences

for the λ repressor were reversed and that O 3 and

3 have the greatest affinity for the repressor. What would

be the likely effect on the mechanism of infection by λ? Explain.

12. Suppose that a mutant C protein of the arabinose operon of E. coli were found that could not bind arabinose.

Predict the results on (a) the level of C protein and (b) the rate of transcription of the genes coding for BAD

enzymes in the presence of cAMP−CAP complex, explaining your reasoning.

BIOLOGIA MOLEKULARNA – Lista 6 – Kontrola ekspresji genów u Prokaryota.