74. Investigation of gene expression in prokaryotes: induction of 􏲙- galactosidase in E. coli

Question 1

I. Background information
1A. What are the features of strong promotors

Answer 1

1. initiation occurs every hour, since the consensus sequence is not altered, but (evolutionary) conserved
2. Affinity for sigma-factor is high = high frequency of RNA polymerase binding

Question 2

I. Background information
1B. What are the features of weak promotors?

Answer 2

• initiation occurs once a week/month, since the consensus sequence is altered
• Affinity for sigma-factor is low = not bind as frequently

Question 3

I. Background information
2. What are the features of constitutive genes?

Answer 3

• housekeeping genes, always turned on
• proteins are continuously produced by the cell

Question 4

I. Background information
3. What are the features of Inducible genes?

Answer 4

• inducible genes are not always are active
• performance depends on environmental conditions and external stimuli that can be
  artificially controlled = so they can be switched “on/off”
  => Repressor or activator proteins recognize and bind specific DNA sequences to control the transcription of a nearby gene

Question 5

I. Background information - Lac operon - negative inducible regulation
4. What are the features of Lac operon - negative inducible regulation?

Answer 5

• It is a sequence of regulatory genes which code for 3 proteins
  required for the breakdown of lactose (disaccharide: galactose + glucose)
• found in the E-coli in the digestive tract of humans, produces the enzyme beta-galactosidase, which makes it possible for us to break down lactose

Question 6

I. Background information - Lac operon - negative inducible regulation
5. Give the coding sequences of protein

Answer 6

• Lac Z - (beta-galactosidase)
• Lac Y - (galactosidase permease)
• Lax A - (galactoside transacetylase)
  _ Lac I gene is the constitutive gene which makes the repressor of lac operon - not a part of the operon itself

Question 7

I. Background information - Lac operon - negative inducible regulation
7. What happen in this Lac Operon in the presence or absence of glucose?

Answer 7

• This operon is usually turned off, since in the presence of glucose, the E-coli do not want to waste energy and time to produce these proteins that digest lactose.
• In the absence of glucose (ex: in starvation), it needs to produce these proteins which can break down lactose.
  =. So, lactose is an alternative for glucose, and only broken down in case the cell needs another energy source.

Question 8

I. Background information - Lac operon - negative inducible regulation
8. How is ß-galactosidase produced?

Answer 8

E.coli can produce the enzyme ß-galactosidase

Question 9

I. Background information - Lac operon - negative inducible regulation
9. What is the role of ß-galactosidase?

Answer 9

• E.coli can produce the enzyme ß-galactosidase, which can catalyze the hydrolysis of the disaccharide lactose to its two monosaccharide components glucose and galactose.
• The monosaccharides can then easily be metabolized by the bacterium

Question 10

I. Background information - function of Lac operon
10. What is the mechanism of Lac operon?

Answer 10

1. Always: repressor tetramer protein is bound to the operator = no transcription
2. Binding of RNA polymerase is enhanced by cAMP-activated CRP (CAP) dimer
3. The presence of Lac repressor di-dimer prevents transcription

Question 11

I. Background information - function of Lac operon
11. How does Lac Operon work in case (+) glucose, (-) lactose?

Answer 11

1) (+) glucose, (-) lactose = no expression of Lac proteins = OPERON OFF
- No lactose present
=> lac repressor binds to lac operator, which overlaps transcription site + no CAP bound

Question 12

I. Background information - function of Lac operon
12. How does Lac Operon work in case (-) glucose, (-) lactose?

Answer 12

(-) glucose, (-) lactose = no expression of Lac proteins = OPERON OFF

Question 13

I. Background information - function of Lac operon
13. How does Lac Operon work in case (+) glucose, (+) lactose?

Answer 13

(+) glucose, (+) lactose = very low expression of Lac proteins = OPERON OFF

Question 14

I. Background information - function of Lac operon
14. How does Lac Operon work in case (-) glucose, (+) lactose?

Answer 14

• (-) glucose, (+) lactose = high expression of Lac proteins = OPERON ON
• Only lactose present
  -> inducer binds to repressor protein
  -> conformational change that
  detaches repressor from operon
• cAMP-CAP complex also greatly increases frequency of transcription initiation
  => activation leads to synthesis of high levels of Lac mRNA

Question 15

II. Experiment
1. What is the background knowledge of the “ Investigation of gene expression in prokaryotes: induction of Beta- galactosidase in E. coli” experiment

Answer 15

• IPTG is a lactose-like molecule with the same effect on the lac repressor as lactose itself. Its advantage over lactose is that it cannot be degraded by beta-galactosidase, so its concentration remains constant.
• Beta-galactosidase can be detected on the basis of its enzymatic activity. We can use the synthetic substrate ONPG (ortho-nitrophenyl-􏲙-galactosidase), a lactose-like molecule, for the determination. Beta-galactosidase will hydrolyze ONPG to galactose and ortho- nitrophenol. Ortho-nitrophenol has a yellow color, which can be quantified by spectrophotometry.

Question 16

II. Experiment
2. What are the 3 steps of enzyme induction?

Answer 16

1) inactivation of the repressor by the inducer
2) mRNA synthesis
3) Translation of mRNAs into proteins

Question 17

II. Experiment
3. What are the effects of different antibiotics in the expression of beta-galactosidase in bacteria?

Answer 17

Induction can be inhibited in each of the steps above. Distinguish between 2 types of inhibitors:
- Rifampicin: inhibitor of bacterial RNA polymerase -> prevents transcription
- Chloramphenicol: directly inhibits bacterial ribosomes -> prevents translation