Name: _________________________________________________ Period:_______________

 

Control of Gene Expression Lab

Background Information: An operon is a group of genes coding for related proteins that are arranged in units that operate together to regulate the expression of genes. An operon consists of an operator, a promoter, regulatory genes and structural genes. The promoter is the part of the DNA to which the RNA polymerase binds before opening the segment of DNA to be transcribed. The regulatory gene codes for a repressor protein that binds to the operator, obstructing the promoter (thus, transcription) of the structural genes which are the segments of DNA that code for specific proteins. There are two types of gene regulation: positive and negative. Negative regulation involves repressor proteins that can be inducible or repressible. Positive regulation involves enhancer proteins.

http://faculty.uca.edu/~johnc/lac%20operon%201.gif

PART A: An example of inducible gene regulation is the lac operon found in the bacteria E. coli. Lactose, milk sugar, is split by the enzyme β-galactosidase. This enzyme is inducible, since it occurs in large quantities only when lactose, the substrate on which is operates, is present. When no lactose is present, the repressor protein binds to the operator and obstructs the promoter so RNA polymerase cannot bind to it and transcription of the structural genes is stopped. In affect, the genes are turned “off”. When lactose is present, the lactose binds to the repressor protein, changing its shape so that the repressor protein can no longer bind to the operator. Lactose is called an inducer. The RNA polymerase can bind to the promoter and transcription of the structural genes occurs. The genes are turned “on”.

 

Directions:

  1. Color the seven different genes of the two lac operons found below the following colors.

pi=promoter for regulatory gene = orange

i = regulatory gene for repressor protein = yellow

plac = promoter for structural genes = red

o = operator = purple

z = structural gene for β-galactosidase = green

y = structural gene for β-galactoside permease = blue

a = structural gene for β-galactoside transacetylase = pink

 

  1. Cut out the repressor protein A and tape it into place (like puzzle pieces) on the operator of the first lac operon.
  2. Cut out the repressor protein A with lactose attached and tape it into place next to the second lac operon.
  3. Answer questions a-f.

Lac Operons:

a)      Text Box: RNA polymerase

 

Repressor A protein can/cannot attach to the operator. (circle one)

 

b)     RNA polymerase can/cannot bind. (circle one)

c)      Transcription of the structural proteins is blocked/proceeds. (circle one)

Text Box: pi
Text Box: i
Text Box: plac
Text Box: o
Text Box: z
Text Box: y
Text Box: a
 

 

 

 


 

 Place repressor protein A here

 

 

 

 

 

 

d)     Repressor protein A with lactose can/cannot attach to the operator. (circle one)

e)      RNA polymerase can/cannot bind. (circle one)

f)       Text Box: RNA polymerase

 

 Transcription of the structural proteins is blocked/proceeds. (circle one)

 

 

 

Text Box: pi
Text Box: i
Text Box: plac
Text Box: o
Text Box: z
Text Box: y
Text Box: a

  

 

 

 


 

Place repressor protein A with

lactose attached here 

 

 

Part B: An example of repressible gene regulation is the tryptophan synthesis operon found in the bacteria E. coli. The enzymes for the amino acid tryptophan are produced continuously in growing cells unless tryptophan is present. If tryptophan is not present, the repressor protein is inactive and transcription of the structural genes occurs. The genes are turned “on”. If tryptophan is present, tryptophan acts as a co-repressor and binds to the repressor protein making it active. The active repressor protein binds to the operator and the production of tryptophan-synthesizing enzymes is stopped. The genes are turned “off”.

Directions:

  1. Color the nine different genes of the two trp operons found below the following colors.

pr=promoter for regulatory gene = blue

r= regulatory gene for repressor protein = pink

ptrp = promoter for structural genes = green

o = operator = yellow

e = structural gene = red

d = structural gene = light blue

c = structural gene = brown

b = structural gene = orange

a = structural gene = purple

 

  1. Cut out the inactive repressor protein B and tape it into place next to the first trp operon.
  2. Cut out the active repressor protein B with the co-repressor tryptophan attached and tape it into place (like puzzle pieces) on the operator of the second trp operon.
  3. Answer questions a-f.

a) Repressor protein B can/cannot attach to the operator. (circle one)

b) RNA polymerase can/cannot bind. (circle one)

Text Box: RNA polymerase

 

c) Transcription of the structural proteins is blocked/proceeds. (circle one)

 

 

 

Text Box: Ptrp
Text Box: o
Text Box: e
Text Box: d
Text Box: c
Text Box: b
Text Box: a
Text Box: r
Text Box: pr

  

 

 


 

Place repressor protein

B here 

 

 

d) Repressor protein B with trp can/cannot attach to the operator. (circle one)

e) RNA polymerase can/cannot bind. (circle one)

Text Box: RNA polymerase

 

f) Transcription of the structural proteins is blocked/proceeds. (circle one)

 

Text Box: Ptrp
Text Box: o
Text Box: e
Text Box: d
Text Box: c
Text Box: b
Text Box: a
Text Box: r
Text Box: pr
 

 

 

 

 

 


 

Place repressor protein B

with tryptophan attached

here 

Part C: The lac operon in E. coli is also an example of a positive control system and is turned on by the cAMP-CAP complex. E.coli’s first choice at every meal is glucose because glucose supplies maximum energy for growth. Therefore, E. coli will only metabolize lactose if concentrations of glucose are low. A small molecule called cyclic AMP (cAMP) acts as an enhancer. The amount of cAMP present in a cell is inversely proportional to the amount of glucose present so no glucose means high levels of cAMP. The cAMP binds to a molecule known as CAP. The cAMP-CAP complex can bind to the promoter and stimulates transcription of the lactose structural genes by helping RNA polymerase bind to the promoter of the lac operon. The genes are turned “on”. With glucose present, there is very little or no cAMP so the cAMP-CAP complex cannot be made.  Without this complex, RNA polymerase cannot bind to the promoter and transcription cannot occur. The genes are turned “off”.

Directions:

  1. Color the seven different genes of the lac operon found below the following colors.

pi=promoter for regulatory gene = orange

i = regulatory gene for repressor protein = yellow

plac = promoter for structural genes = red

o = operator = purple

z = structural gene for β-galactosidase = green

y = structural gene for β-galactoside permease = blue

a = structural gene for β-galactoside transacetylase = pink

 

  1. Cut out the cAMP-CAP complex and tape it into place (like puzzle pieces) on the promotor of the first lac operon.
  2. Answer questions a-c.

a)      cAMP-CAP complex can/cannot attach to the promoter. (circle one)

b)     RNA polymerase can/cannot bind. (circle one)

c)      Text Box: RNA polymerase

 

 Transcription of the structural proteins is blocked/proceeds. (circle one)

 

 

 

Text Box: pi
Text Box: i
Text Box: plac
Text Box: o
Text Box: z
Text Box: y
Text Box: a

  

 

 


 

Place cAMP-CAP

complex here

 

Questions:

1.      What is an operon?

 

2.      What is the function of the promoter?

 

3.      What is the function of the operator?

 

4.      If repressor protein A in Part A is attached to the operator, are the genes “on” or “off”?

5.      If the genes are “off”, what process does NOT happen?

 

6.      What happens to the repressor protein A if lactose is present?

 

7.      Are the genes turned “on” or “off” if lactose is present?

8.      If the genes are “on”, what process does happen?

 

9.      What type of negative gene regulation occurs in the lac operon in Part A?

 

10.  What activates the repressor protein B?

 

11.  When the repressor protein B is activated, are the genes “on” or “off”?

12.  In Part B, what turns the tryptophan operon “on”?

 

13.  What type of negative gene regulation occurs in the trp operon in Part B?

 

14.  What acts as an enhancer for the lac operon in Part C?

 

15.  What is the role of the cAMP-CAP complex?