Benjamin A.

Pierce

GENETICS
A Conceptual Approach
FIFTH EDITION

CHAPTER 16
Control of Gene Expression in Prokaryotes

© 2014 W. H. Freeman and Company

E. coli
 16.1 The Regulation of Gene Expression is
Critical for All Organisms

• Genes and Regulatory Elements

•

• Levels of Gene Regulation

•

• DNA-Binding Proteins
 Genes and Regulatory Elements

• Structural genes: encoding proteins

•

• Regulatory genes: encoding products that interact

with other sequences and affect the transcription and
translation of these sequences/genes (transcription
factors)
•

• Regulatory elements: DNA sequences that are not

transcribed but play a role in regulating other
nucleotide sequences/genes (consensus seq)
 Genes and Regulatory Elements

• Positive control: stimulate gene expression (on)

•

• Negative control: inhibit gene expression (off)

•

• Constitutive expression: continuously expressed

under normal cellular conditions
Levels of Gene Regulation

(chromatin) - accessibility

- on, off, constitutive, where/when

- 5’ Cap, poly-A tail, splicing etc.

- how much mRNA/how fast degraded

- how fast protein is synthesized

- affect function of the protein (active or

not active)
 DNA-Binding Proteins

• DNA-binding Domains: ~ 60 - 90 amino acids, responsible

for binding to DNA, forming hydrogen bonds with DNA
(usually just part of the protein called the DNA-binding
domain). These proteins can have different types of domains
that bind other regulatory proteins, but typically only have 1
DNA-binding domain.
•

• Motif: within the binding domain, a simple structure that fits

into the major groove of the DNA
• Distinctive types of DNA-binding proteins based on the motif
(that is, any given DNA-binding protein may have a particular
type of motif that recognizes and binds a specific DNA
sequence)
 Different types of motifs

more common in more common in

eukaryotes eukaryotes
more common in bacteria

For example: there are numerous Zinc finger DNA-binding

proteins that (A) bind to different regulatory proteins and (B)
recognize different sets of DNA sequences (DNA motifs)
 Concept Check 1

How do amino acids in DNA-binding proteins interact

with DNA?
a.

a. By forming covalent bonds with DNA base

b.

c. By forming hydrogen bonds with DNA base

d.

e. By forming covalent bonds with sugars

Hydrogen bonds are relatively weak, this allows the DNA-binding proteins to come off
and on easily and compete with other regulatory proteins (ie. dynamic)
16.2 Operons Control Transcription in Bacterial
Cells
Operon: a group of bacterial structural genes that are
transcribed together (along with their promoter and
additional sequences that control transcription)

• Operon Structure
• Negative and Positive Control; Inducible and Repressible
Operons
• The lac Operon of E. coli
• lac Mutations
• Positive Control and Catabolite Repression
• The trp Operon of E. coli
 Operon Structure

• Operon: promoter + additional sequences that control

transcription (operator) + structural genes
• Regulator gene: DNA sequence encoding products that
affect the operon function, but are not part of the operon
 Concept Check 2

What is the difference between a structural gene and a

regulator gene?
a.

a. Structural genes are transcribed into mRNA, but

regulator genes are not.
b. Structural genes have complex structures; regulator
genes have simple structure.
c. Structural genes encode proteins that function in the
structure of the cell; regulator genes carry out metabolic
reactions.
d. Structural genes encode proteins; regulator genes control
the transcription of structural genes.
 Negative and Positive Control; Inducible and
Repressible Operons
• Negative control: regulatory protein is a repressor of
transcription
•

• Positive control: regulatory protein is an activator of

transcription

• Inducible operons: Transcription is usually off and needs

to be turned on.

• Repressible operons: Transcription is normally on and

needs to be turned off.
 Negative and Positive Control; Inducible and
Repressible Operons

• Negative inducible operons: The control at the operator

site is negative (repressor). Molecule binding is to the
operator, inhibiting transcription. Such operons are
usually off and need to be turned on, so the transcription
is inducible.
- Inducer: small molecule that turns on the
transcription.

Negative inducible operons

repressor

transcription off, needs to be turned ON

Negative inducible operons

Proteins which change

shape upon binding with
to another molecule are
allosteric proteins

Economical, the
enzymes are
synthesized only when
their substrate (V) is
available
 Negative and Positive Control: Inducible and
Repressible Operons

• Negative repressible operons: The control at the operator

site is negative (repressor). But such transcription is
usually on and needs to be turned off, so the transcription is
repressible.
- Corepressor: a small molecule that binds to the
repressor and makes it capable of binding to the operator to
turn off transcription.

Negative repressible operons

repressor

Transcription on, needs to be turned OFF

Negative repressible operons
 Negative and Positive Control: Inducible and
Repressible Operons
• Positive inducible operon: transcription is normally turned
off because the regulator protein (an activator) is produced
in an inactive form. Transcription takes place when an
inducer is attached to the regulatory protein, rendering the
regulator active.
• Positive repressible operon: the regulatory protein is
produced in a form that readily binds to DNA, meaning that
transcription normally takes place and has to be repressed.
A substance must bind to the regulatory protein to render it
inactive
•

- In positive control the regulator protein is an activator

 Positive and negative control in
inducible and repressible operons

When the enzyme is low

(blue diamond), the
precursor (purple circle)
is high, thus turning
transcription on to make
the enzyme. The opposite
is true, so the
system/balance is
economical.

Once the end product

(blue diamond) is in
abundance, it binds to the
regulatory protein to
render it inactive, thus
turning off transcription.
 The lac Operon of E. coli

• A negative inducible operon

• Lactose metabolism
• Regulation of the lac operon
• Inducer: allolactose
•

• lacI: repressor encoding gene

• lacP: operon promoter
• lacO: operon operator
 The lac Operon of E. coli

• Structural genes
•

• lacZ: encoding β-galactosidases

• lacY: encoding permease
• lacA: encoding transacetylase
•

• The repression of the lac operon never completely

shuts down transcription – there are always a few
molecules of the structural genes
Lactose Metabolism

(lacY)

(lacZ)

(inducer)
Regulation of the Lac Operon (negative inducible operon)
François Jacob
and Jacques
Monod, 1961

coordinate induction:
simultaneous synthesis
of structural proteins
stimulated by a specific
molecule, the inducer.
 The lac operator overlaps the 5’ end of the lacZ gene

Concept Check 4

In the presence of allolactose, the lac repressor .

a.

a. binds to the operator

b. binds to the promoter
c. cannot bind to the operator
d. binds to the regulator gene
 If the lac operon is repressed and
no permease is being produced,
how does lactose get into the cell
to inactivate the repressor and
turn on transcription?

Furthermore, the inducer is

actually allolactose, which must
be produced from lactose by β-
galactosidase. If β-galactosidase
production is repressed, how can
lactose metabolism be induced?

Repression never completely shuts down. Even with active repressor bound to the
operator, there is a low level of transcription and a few molecules of β-galactosidase,
permease, and transacetylase are synthesized.
 lac Mutations

In order to understand the structure and function of the Lac operon, Jacob
and Monod used mutations that affected lactose metabolism.
• Partial diploid: full bacterial chromosome plus an
extra piece of DNA on F plasmid F’

• lacZ+lacY−
contains a lac operon (also a lac operon on
the bacterial chromosome)
• Structural gene mutations: affect the structure of the
lacZ lacY - +

enzymes (β-galactosidase and permease), but not the

regulation of their synthesis
•

- lacZ+lacY−/lacZ−lacY+ produce fully functional

β-galactosidase and permease.
 lac Mutations
•

• Regulator gene mutations: lacI− leads to constitutive

transcription of the structure genes. (normally lacI =
repressor encoding gene)
•

• lacI+ is dominant over lacI− and is trans acting. A single

copy of lacI+ brings about normal regulation of lac
operon (lacI+/lacI−)
• Example: lacI+lacZ−/lacI−lacZ+ produce fully
functional β-galactosidase.
 Regulator gene mutation: lacl-

Chromosome: lacI+lacZ−

F plasmid: lacI-lacZ+

Chromosome: lacI+lacZ−

F plasmid: lacI-lacZ+

lacl is trans-acting: able to control the

expression of genes on other DNA
molecules (chromosome and F plasmid)
 Regulator gene mutation: laclS
Both the normal lac repressor and the lac super-repressor can
bind to the operator and inhibit transcription

Chromosome:

F plasmid:
This would normally lead to β-galactosidase
production, except the supperrepressor inhibits
production

Unlike lacl-, laclS can bind to operator whether lactose is present or not
 lac Mutations

• Operator mutations: altered the sequence of DNA at the

operator so that the repressor protein is no longer able to
bind. lacOc: C = constitutive
•

• lacOc is dominant over lacO+, which is cis-acting.

•

•
able to control the expression of genes
only +when on the+same piece of DNA
• lacI+lacO+lacZ /lacI lacO lacZ produce fully
− c

functional β-galactosidase constitutively.

 Mutations in lacO are constitutive and cis-acting (DNA)

Chromosome:

F plasmid:

constitutive

lacOc only affecting lacZ+ lacOc only affecting lacZ-

 lac Mutations
•

• Promoter mutations: lacP−: cis-acting

• lacI+lacP−lacZ+/lacI+lacP+lacZ− fails to produce function
β-galactosidase (cis acting = DNA) Chromosome: lacI+lacP+lacZ-
•

Plasmid: lacI+lacP-lacZ+

X-
•

• vs
• lacI+lacZ−/lacI−lacZ+ produce fully functional β-galactosidase
• Look at “worked problem” page 474
 Positive Control and Catabolite Repression

• Catabolite repression: E. coli prefer to use glucose when

available because it takes less energy to metabolize than
other sugars. When using glucose, bacteria repress the
metabolism of other sugars (including lactose).
•

• The Lac operon is only transcribed when lactose is

present AND glucose is absent
 Positive Control and Catabolite Repression

• Catabolite repression happens via a positive control

mechanism. The positive effect is activated by catabolite
activator protein (CAP). CAP is bound by cyclic AMP
(cAMP); this CAP–cAMP complex binds to a site slightly
upstream from the lac gene promoter and increases
transcriptional efficiency
- cAMP: adenosine-3′, 5′-cyclic monophosphate
(a modified nucleotide important for cellular signaling)
• The concentration of cAMP is inversely proportional to the
level of available glucose (high cAMP = low glucose).
 Catabolite Repression
and lactose is available

~ 20 operons are affected by CAP:

this is how glucose regulates
catabolite repression
How does cAMP-CAP enhance transcription?

Bends DNA
which in turn
facilitates RNA
polymerase
binding to the
promoter
 Concept Check 5

What is the effect of high levels of glucose on the

lac operon?
a.

a. Transcription is stimulated.
b. Little transcription takes place.
c. Transcription is not affected.
d. Transcription may be stimulated or inhibited, depending
on the levels of lactose.
 The trp Operon of E. coli

•
A negative repressible operon- transcription is
normally turned on and must be repressed.
•

•
Five structural genes
•

• - trpE, trpD, trpC, trpB, and trpA—five enzymes

together convert chorismate to tryptophan.
The trp operon – negative repressible

trpR

trpR
 16.3 Some Operons Regulate Transcription
Through Attenuation, the Premature
Termination of Transcription
• Attenuation: affects the continuation of
transcription, not its initiation. This action
terminates the transcription before it reaches the
structural genes.
•

• Attenuator – terminates transcription

•

• Antiterminator – prevents termination

 Attenuation in the trp Operon of E. coli

• Four regions of the long 5′ UTR (leader) region of trpE

mRNA
•

• When tryptophan is high, region 1 binds to region 2,

which leads to the binding of region 3 and region 4,
terminating transcription prematurely.
•

• When tryptophan is low, region 2 binds to region 3,

which prevents the binding of region 3 and region 4,
and transcription continues.
 The 5’ UTR of the trp operon mRNA regulates attenuation

5’ UTR not normally translated, but in the trp operon it is (exception to the rule)

region 1 is complementary to region 2 region

2 is complementary to region 3 region 3 is
complementary to region 4

The 3+4 hairpin plus string of U’s

is similar in structure to the
bacterial intrinsic terminator

attenuator antiterminator
 How do the different secondary structures form?
* Remember,
transcription and
translation can
occur nearly
simultaneously in
bacteria

antiterminator
translation stalls
so transcription
proceeds ahead
The small protein produced is unstable and quickly degraded.
Only made for the purpose of attenuation

Trp

attenuation

* Because tryptophan is abundant, translation can keep up with transcription (coupled)

 Concept Check 7

Attenuation results when which regions of the 5′ UTR

region pair?
a.

a. 1 and 3
b. 2 and 3
c. 2 and 4
d. 3 and 4
 Why Does Attenuation Take Place in the trp Operon?

- Repression is never complete; transcription reduced ~ 70-fold, while attenuation

reduces another ~ 8-fold to reduce more than 600-fold
-

- attenuation and repression respond to different signals: repression responds to the

cellular levels of tryptophan, whereas attenuation responds to the number of tRNAs
charged with tryptophan.
* Play animation 16.2
16.4 RNA Molecules Control the Expression of
Some Bacterial Genes

• Antisense RNA: complementary to targeted

partial sequence of mRNA
• Riboswitches: molecules influence the
formation of secondary structures in mRNA
• Ribozymes: mRNA molecules with catalytic
activity
Riboswithces