Control of Gene Expression in Bacteria

1
Chapter 14

• Control of Gene Expression in Bacteria

2
Chapter 14
3
Gene Regulation Mechanisms in Bacteria

• Bacteria express only a subset of their genes at
  any given time.
• Expression of all genes constitutively in
  bacteria would be energetically inefficient.

4
Gene Regulation Mechanisms in Bacteria

• Bacteria express only a subset of their genes at
  any given time.
• The genes that are expressed are essential for
  dealingwith the current environmental
  conditions, such as thetype of available food
  source.

5
Gene Regulation Mechanisms in Bacteria

• Regulation of gene expression can occur at
  several levels
• Transcriptional regulation no mRNA is made.
• Translational regulation control of whether or
  how fast an mRNA is translated.
• Post-translational regulation a protein is made
  in an inactive form and later is activated.
  (Fig. 14.1)

6
Figure 14.1
Transcriptional control
Translational control
Post-translational control
Lifespan of mRNA
Protein activation (by chemical
modification)
Protein
Onset of transcription
Translation rate
Feedback inhibition (protein inhibits
transcription of its own gene)
Ribosome
mRNA
DNA
RNA polymerase
7
Gene Regulation Mechanisms in Bacteria

• Case study of the regulation of the lactose
  operon in E. coli
• E. coli utilizes glucose if it is available, but
  can metabolizeother sugars if glucose is absent.
  (Fig. 14.2)

8
Figure 14.2
Glucose Lactose
Glucose Lactose
Glucose Lactose
13
11
31
Food source
Second period of rapid growth with lactose as
food source
70
60
29.5
50
14.0
40
43.5
Relative density of cells
30
20
26.5
Initial period of rapid growth with glucose as
food source
39.0
13.5
10
0
0
2
3
4
5
0
1
3
4
5
6
0
1
2
1
2
3
4
5
6
7
Time (hours)
9
Figure 14.3
REPLICA PLATING
Velvet-covered block
Mutant E. coli cells
Master plate
One E. coli cell grows to produce a single colony.
Many sugars
Many sugars
Absence of colony
Absence of colony
Many sugars
Many sugars
Lactose only
Lactose only
Many sugars
Many sugars
Lactose only
Lactose only
Master plate
Master plate
Replica plate
Replica plate
1. Grow a master plate of E. coli colonies on
complete medium (contains many sugars as energy
source).
2. Press velvet-covered block against master
plate. Some cells transfer from plate to block.
3. Press block against a replica plate containing
only lactose as an energy source. Location of
cells on master plate and replica plate are an
exact match.
4. After incubation, cells that can use lactose
as energy source grow into colonies. Pick cells
on master plate that are not on replica plate.
These cells are deficient in lactose metabolism.
10
Figure 14.3
REPLICA PLATING
Mutant E. coli cells
Velvet-covered block
Master plate
One E. coli cell grows to produce a single colony.
Absence of colony
Many sugars
Many sugars
Absence of colony
Many sugars
Many sugars
Lactose only
Lactose only
Many sugars
Many sugars
Lactose only
Lactose only
Master plate
Master plate
Replica plate
Replica plate
1. Grow a master plate of E. coli colonies on
complete medium (contains many sugars as energy
source).
2. Press velvet-covered block against master
plate. Some cells transfer from plate to block.
3. Press block against a replica plate containing
only lactose as an energy source. Location of
cells on master plate and replica plate are an
exact match.
4. After incubation, cells that can use lactose
as energy source grow into colonies. Pick cells
on master plate that are not on replica plate.
These cells are deficient in lactose metabolism.
11
Gene Regulation Mechanisms in Bacteria

• Case study of the regulation of the lactose
  operon in E. coli
• Genes that encode enzymes needed to break other
  sugarsdown are negatively regulated.
• Example enzymes required to metabolize lactose
  are only synthesized if glucose is depleted and
  lactose is available.

12
Gene Regulation Mechanisms in Bacteria

• Case study of the regulation of the lactose
  operon in E. coli
• Genes that encode enzymes needed to break other
  sugarsdown are negatively regulated.
• In the absence of lactose, transcription of the
  genes that encode these enzymes is repressed. How
  does this occur?

13
Gene Regulation Mechanisms in Bacteria

• Case study of the regulation of the lactose
  operon in E. coli
• Jacob and Monod screened mutants to identify
  genetic loci involved in lactose metabolism.
• All the loci required for lactose metabolism are
  groupedtogether into an operon.
• The lacZ locus encodes ?-galactosidase enzyme,
  which breaksdown lactose.
• The lacY locus encodes galactosidase permease, a
  transportprotein for lactose.
• The function of the lacA locus is unknown.
• The lacI locus encodes a repressor that blocks
  transcriptionof the lac operon. (Fig. 14.4,14.8a)

14
Figure 14.4
Cleaves lactoseto glucose and galactose
Membrane transport protein-imports lactose
Regulatory function
Regulatory protein
Galactosidase permease
ß-galactosidase
Lacl
LacY
LacZ
Section of E. coli chromosome
lacl
lacZ
lacY
Observations about regulation of lacZ and lacY
Glucose
(1) Lacl protein and glucose shut down
transcription of lacZ and lacY
Lactose
E. coli
Galactose
Galactosidase permease
(2) Lactose induces transcription of lacZ and lacY
Chromosome
ß-galactosidase
15
Figure 14.8a
Lac operon
Lac operon
lacl promoter
lacl
Promoter
Operator
lacZ
lacY
lacA
16
Gene Regulation Mechanisms in Bacteria

• Case study of the regulation of the lactose
  operon in E. coli
• Repression and induction of the lactose operon.
• The lac operon is under negative regulation, i.e.
  , normally, transcription is repressed.

17
Gene Regulation Mechanisms in Bacteria

• Case study of the regulation of the lactose
  operon in E. coli
• Repression and induction of the lactose operon.
• Glucose represses transcription of the lac
  operon.
• Glucose inhibits cAMP synthesis in the cells.
• At low cAMP levels, no cAMP is available to bind
  CAP.
• Unless CAP is bound to the CAP site in the
  promoter, no transcription occurs. (Fig. 14.4,
  14.9b)

18
Figure 14.4
Cleaves lactoseto glucose and galactose
Membrane transport protein-imports lactose
Regulatory function
Regulatory protein
Galactosidase permease
ß-galactosidase
Lacl
LacY
LacZ
Section of E. coli chromosome
lacl
lacZ
lacY
Observations about regulation of lacZ and lacY
Glucose
(1) Lacl protein and glucose shut down
transcription of lacZ and lacY
Lactose
E. coli
Galactose
Galactosidase permease
(2) Lactose induces transcription of lacZ and lacY
Chromosome
b-galactosidase
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Box 14.1, Figure 1
Experimental evidence that bacteria can transfer
genes to other bacteria
1. Grow E. coli cells in a medium that
contains all the amino acids that they cannot
produce themselves.
STRAIN A STRAIN B
STRAIN A
STRAIN B
Both genotypes
Medium contains leucine, threonine, thiamine,
cysteine, phenylalanine, and biotin
Medium contains cysteine, phenylalanine, and
biotin
Medium contains leucine, threonine, and thiamine
2. Plate cells into minimal medium (contains no
amino acids). Colonies will only form if the
bacteria can produce all their own amino acids.
No growth
No growth
Controls Plate cells onto minimal medium
(contains no amino acids)
20
Box 14.1, Figure 2
STRAIN A
STRAIN B
Transfer cells
Transfer cells
Filter allows DNA to pass, but not cells
No growth in minimal medium
No growth in minimal medium
21
Box 14.1, Figure 3
CONJUGATION
22
Figure 14.6a, upper
THE PAJAMo EXPERIMENT
1. Isolate donor bacterial cells with normal
copies of lacl and lacZ genes and sensitivity
to streptomycin. (These cells are killed by
streptomycin.)

Donor cell

s
Genotype
Phenotype
Normal repressor
lacl
ß-galactosidase
lacZ
str s
Sensitive to streptomycin
2. Isolate recipient bacterial cells with lacl
and lacZ genes and resistance to streptomycin.
(These cells are not killed by streptomycin.)


Recipient cell
r
Genotype
Phenotype
lacl
No repressor
lacZ
No ß-galactosidase
str r
Resistant to streptomycin
23
Figure 14.6a, lower

3. Mix populations of donor cells and recipient
cells and allow conjugation to proceed. Begin
withdrawing cell samples at regular intervals to
measure ß-galactosidase activity.
Donor cell

s
Conjugation


Recipient cell


r
4. After 1.5 hours (when conjugation is
complete), kill all donor cells with
streptomycin. Continue measuring
ß-galactosidase activity in recipient cells.

Dead donor cell

s




r
24
Figure 14.6b
Initial results
10
Amount of ß-galactosidase (enzyme activity/ mL)
5
0
0
2
1
3
4
5
6
7
Hours after start of conjugation
25
Figure 14.6c
20
Results when IPTG (an inducer) was added 2
hours after start of conjugation
15
Amount of ß-galactosidase (enzyme activity/ mL)
10
5
0
0
1
2
3
4
5
6
7
Hours after start of conjugation
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Figure 14.9a
Catabolism
Large reactant molecule
Small product molecule
Small product molecule
Enzyme
27
Figure 14.9b
Catabolite repression of the lac operon
HOCH2
HOCH2
HOCH2
OH
HO
O
HO
O
OH
OH
O
O
OH
OH
OH
OH
OH
HO
ß-galactosidase
O
OH
OH
OH
HOCH2
Glucose
Galactose
Lactose
Presence of glucose suppresses production of
enzyme
28
Gene Regulation Mechanisms in Bacteria

• Case study of the regulation of the lactose
  operon in E. coli
• Repression and induction of the lactose operon.
• The product of the lacI locus is a repressor that
  blockstranscription of the lac operon.
• The repressor binds to DNA at sites called
  operators. (Fig. 14.13a,b)

29
Figure 14.13a,b
Multiple binding sites for repressor
Sequence at 01
30
Gene Regulation Mechanisms in Bacteria

• Case study of the regulation of the lactose
  operon in E. coli
• Repression and induction of the lactose operon.
• If the operators are bound by a repressor, RNA
  polymerase is blocked from transcribing the
  genes. (Fig. 14.5a, 14.7a)

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Figure 14.5a
Repressor present (normal lacl gene). No
transcription.
NO TRANSCRIPTION
Repressor molecule synthesized
lacl
lacZ
lacY
32
Figure 14.5b
No repressor present (mutant lacl gene).
Transcription proceeds.
TRANSCRIPTION BEGINS
No functional repressor synthesized
mRNA
b-galactosidase
Permease
lacl _
lacZ
lacY
33
Figure 14.7a
When no lactose is present, the repressor binds
to DNA and blocks transcription.
NO TRANSCRIPTION
Functional repressor
lacl
lacZ
lacY
RNA polymerase blocked
Operator (binding site for repressor)
34
Gene Regulation Mechanisms in Bacteria

• Case study of the regulation of the lactose
  operon in E. coli
• Repression and induction of the lactose operon.
• Lactose induces transcription of the lac operon.
• Lactose enters the cell and is converted to
  allolactose.
• Allolactose binds to the repressor and causes it
  to release the operator. (Fig. 14.5c, 14.7b)
• RNA polymerase transcribes the gene, if CAP is
  bound to the CAP site. (Fig. 14.8b, 14.10a-c,
  14.11)

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Figure 14.5c
Repressor plus lactose (an inducer) present.
Transcription proceeds.
Lactose
TRANSCRIPTION BEGINS
?-galactosidase
Permease
mRNA
Pepressor
lacl
lacZ
lacY
36
Figure 14.7b
When lactose is present, it binds to repressor.
The inducer-repressor complex falls off DNA,
allowing transcription to proceed.
mRNA
lacZ message
Lactose
TRANSCRIPTION
lacl
lacZ
Operator
lacY
Inducer-repressor complex
37
Figure 14.8a
Lac operon
Lac operon
lacl promoter
lacl
Promoter
Operator
lacZ
lacY
lacA
38
Figure 14.8b
Operons produce mRNAs that code for functionally
related proteins.
"Polycistronic" mRNA
lacZ message
lacY message
RNA polymerase binds to promoter
lacA message
lacl promoter
lacl
Promoter
Operator
lacZ
lacY
lacA
39
Figure 14.10a
40
Figure 14.10b
41
Figure 14.10c
42
Figure 14.11
43
Introduction to DNA Binding Proteins

• Proteins that bind to DNA share similarity in the
  structure of their DNA-binding regions.
• Many DNA binding proteins, such as lac repressor,
  have a helix-turn-helix motif which fits into
  the major groove of a DNA molecule. (Fig.
  14.14a,b)

44
Figure 14.14a,b,c
(a)
(b)
(c)
45
Introduction to DNA Binding Proteins

• Proteins that bind to DNA share similarity in the
  structure of their DNA-binding regions.
• Binding of an inducer to the lac repressor causes
  it to release the operator DNA because it alters
  the conformation of the helix-turn-helix motif.
  (Fig. 14.15a-c)

46
Figure 14.15a
47
Figure 14.15b
48
Figure 14.15c
49
Introduction to DNA Binding Proteins

• Information about regulation of the expression of
  genetic loci may help to combat diseases.
• Virulent bacterial strains have genes that encode
  the abilityto infect and produce disease.

50
Introduction to DNA Binding Proteins

• Information about regulation of the expression of
  genetic loci may help to combat diseases.
• Knowledge of how the expression of these genes is
  controlled and regulated may provide insights
  into blocking the development of the disease.

51
Figure 14.12
Glucose HIGH Lactose LOW
Glucose HIGH Lactose HIGH
Glucose LOW Lactose HIGH
Glucose LOW Lactose LOW
52
Box 14.2, Figure 1
When tryptophan is absent, transcription occurs.
RNA polymerase
Leader
5 coding loci
Promoter
When tryptophan is present, transcription is
blocked.
Tryptophan
Repressor
53
Box 14.2, Figure 2
leading to formation of stem-and-loop
structure that inhibits RNA polymerase and
terminates transcription.
Ribosomes translates mRNA rapidly when
tryptophan is abundant,
54
Box 14.3, Figure 1
DNA FOOTPRINTING
1. Generate fragments from the DNA region of
interest, such as the lac operon of E. coli.
Attach radioactive label.
Radioactive atom
Repressor protein present
2. Divide fragments into two treatments, with or
without repressor protein. The repressor will
bind to the operator.
No repressor protein present
3. Cut fragments with nuclease to
produce fragments of different lengths. Repressor
protects DNA from nuclease cleavage.
4. Load fragments into two lanes in a gel. Sort
by size via electrophoresis. (The fragments with
a radioactive atom will be visible.
Largest fragments
Footprint No cuts occurred in the DNA region
protected by the repressor. This region must be
the operator.
The dideoxy sequencing reaction can be used to
determine the sequence of the footprint.
Smallest fragments
55
Applying Ideas, Question 4