Lecture PowerPoint to accompany

Molecular Biology
Fifth Edition

Robert F. Weaver
Chapter 7
Operons:
Fine Control of
Bacterial Transcription
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
 COLOR
ART & PHOTOS
 General Rules for Gene Regulation

1. Synthesis occurs only when needed

2. Two pathways
Catabolic (breakdown) systems are inducible by substrates
Anabolic (biosynthetic) systems are repressible by products

3. Most systems have more than one regulatory mechanisims

7-4
 7.1 The lac Operon
• The lac operon was the first operon
discovered (Jacob & Monod, 1940)
• Contains 3 genes coding for E. coli
proteins that permit the bacteria to use the
sugar lactose
– Galactoside permease which transports
lactose into the cells
− b-galactosidase cuts the lactose into
galactose and glucose
– Galactoside transacetylase whose function is
unclear
7-5
Fig. 7.1
Diauxic growth of E. coli grown on a medium containing both glucose and lactose
Fig. 7.2

1’ 4’

β-glycosidic bond (β-1.4)

Sugar + non-sugar
Glycon aglycon
Aspirin salicin
 Genes of the lac Operon
• Genes are grouped:
– lacZ = b-galactosidase
– lacY = galactoside permease
– lacA = galactoside transacetylase
• All 3 genes are transcribed together producing 1
mRNA, a polycistronic message that starts from
a single promoter
– Each cistron, or gene, has its own ribosome binding
site (Shine-Dalgarno)
– Each cistron can be translated by separate ribosomes
that bind independently of each other

* Expression is somewhat leaky – a low basal level of

lac operon products is always present. 7-8
 Control of the lac Operon

• The lac operon is tightly controlled, using 2

types of control
– Negative control, like the brake of a car,
must remove the repressor from the operator
- the “brake” is a protein called the lac
repressor
– Positive control, like the accelerator pedal of
a car, an activator, additional positive factor
responds to low glucose by stimulating
transcription of the lac operon

7-9
 Negative Control of the lac
Operon
• Negative control indicates that the operon
is turned on unless something turns it off
and stops it
• The off-regulation is done by the lac
repressor
– Product of the lacI gene
– Tetramer of 4 identical polypeptides
– Binds the operator just right of promoter

7-10
 lac Repressor
• When repressor binds the operator,
operon is repressed
– Operator and promoter are contiguous
– Repressor bound to operator prevents
RNA polymerase from binding to the
promoter
• As long as no lactose is available, lac
operon is repressed

7-11
Negative Control of the lac Operon

Allosteric effect
Dissociation 유도

7-12
 Inducer of the lac Operon
•The repressor is an allosteric protein
– Binding of one molecule to the protein changes shape of
a remote site on that protein
– Altering its interaction with a second molecule
•Inducer (one molecule) of lac operon binds the
repressor
– Causing the repressor to change conformation that
favors release from the operator (the second molecule)
•The inducer is allolactose, an alternative form of
lactose
7-13
 Inducer of the lac Operon
• The inducer of the lac operon binds the repressor
• The inducer is allolactose, an alternative form of
lactose

β-galactoside
β-galactoside
7-14
 Discovery of the Operon
During the 1940s and 1950s, Jacob and
Monod studied the metabolism of lactose by
E. coli(1965년 노벨생리의학상 수상)
•Three enzyme activities / three genes were
induced together by galactosides
• Constitutive mutants need no induction,
genes are active all the time
• Created merodiploids or partial diploid
bacteria carrying both wild-type (inducible)
and constitutive alleles
7-15
 Discovery of the Operon
• Using merodiploids or partial diploid bacteria
carrying both wild-type and constitutive alleles
distinctions could be made by determining whether
the mutation was dominant or recessive
• Because the repressor gene produces a repressor
protein that can diffuse throughout the nucleus, it can
bind to both operators in a merodiploid and is called a
trans-acting gene because it can act on loci on both
DNA molecules
• Because an operator controls only the operon on
the same DNA molecule it is called a cis-acting gene
7-16
Fig. 7.5
Fig. 7.6
 Effects of Regulatory Mutations:
Wild-type and Mutated Repressor

wt allele

inducible

mutant allele

7-19
 Mapping the lac operon
• cis/trans test:
– “cis dominance” means the gene is dominant
only when cis, i.e. located on the same piece
of DNA
– i.e. does not operate via a diffusible
intermediate
• e.g. operator vs. repressor:
O- dominant only when cis
i+ dominant cis or trans

7-20
Effects of Regulatory Mutations:
Wild-type and Mutated Operator with
Defective Binding

is-
Operator-
constitutive
mutant
nonrespressible

Constitutive하게 발현

7-21
Effects of Regulatory Mutations:
Wild-type vs Mutated repressor binding irreversibly

R can not recognize the inducer and

bind to the inducer

Irreversible binding (inducer 가

repressor를 떨어뜨리지 못함 물론 respressible

R can not bind to the operator

spoiler

Constitutive and
dominant
7-22
Repressor-Operator Interactions
• Using a filter-binding assay, the lac
repressor binds to lac operator
• A genetically defined constitutive lac
operator (Oc) has lower than normal
affinity for the lac repressor (Figure 7.6)
• Sites defined by two methods as the
operator are in fact the same

7-23
Fig. 7.7

Assaying the binding between lac operator and lac repressor inducer
R
32P

DNA
P O

DNA
Isopropyl β-D-1-thiogalactopyran
R

Affinity 차이

1mM IPTG prevents repressor-operator binding.

Fig. 7.8

Oc lac operator binds repressor with lower affinity than the wt operator

R
32P

-- IPTG DNA
P Owt

P Oc

Lower affinity -- more repressor needed

 The Mechanism of Repression
• The repressor does not block access by
RNA polymerase to the lac promoter
• Polymerase and repressor can bind
together to the lac promoter
• Polymerase-promoter complex is in
equilibrium with free polymerase and
promoter

7-26
 Hypotheses
for mechanisms of repression

• Repression by steric hindrance

• Inhibition of transition to open complex
• Inhibition of promoter clearance
• Anti-activation
• Anti-sigma factors

가장 그럴듯한 설명: Repressor가 RNA Poly의 접근, 결합을 허용하고, open

promoter complex까지 형성하도록 하지만, elongation state로의 전이를 억제
7-27
Fig. 7.9

RNA polymerase forms an open promoter complex with the lac promoter
even in the presence of lac repressor in vitro.

Thus, repressor did not prevent polymerase

from binding and forming an open promoter
complex.
lac Repressor and Dissociation of
RNA Polymerase From Promoter
• Without competitor, Effect of lac repressor on reassociation of
dissociated polymerase RNA polymerase with the lac promoter.
returns to promoter
• Heparin and repressor
prevent reassociation of
polymerase and promoter
• Repressor prevents
reassociation by binding to
the operator adjacent to the
promoter
• This blocks access to the
promoter by RNA
polymerase
7-29
 Mechanism Summary
• Two hypotheses of mechanism for
repression of the lac operon
– RNA polymerase can bind to lac promoter in
presence of repressor
• Repressor will inhibit transition from abortive
transcription to processive transcription
– Repressor, by binding to operator, blocks
access by the polymerase to adjacent
promoter (original hypothesis)

7-30
 lac Operators
• There are three lac operators
– The major lac operator lies adjacent to
promoter
– Two auxiliary lac operators - one upstream
and the other downstream
• All three operators are required for
optimum repression
• The major operator produces only a
modest amount of repression
7-31
Fig. 7.11

Three lac operators

CAP: positive regulator

Functional lac repressor is a homotetramer
• Each dimer binds to a distinct
DNA sequence at –82 and +11
respective to transcription start
O3 O1
site
+11 • This results in DNA looping,
-82
preventing the DNA polymerase
from binding to –35 and –10
sequences
-35

-10

-82 +11

lac
repressor 7-33
Fig. 7.12

Effect of mutations in the three lac operators

Lac Z (operator)

92bp 401bp
Major(classical)

Lac I

Cooperativity among operators

Fig. 7.13

Structure of lac repressor tetramer bound to two

operator fragments

O3 O1

2 DNA binding domains/ dimer

Major groove에 결합
각 dimer는 서로 떨어진(O1, O3) operator에 결합
7
-
3
6
Functional lac repressor is a homotetramer
• Each dimer binds to a distinct DNA
sequence at –82 and +11 respective to
transcription start site
O3 O1 • This results in DNA looping,
+11 preventing the DNA polymerase from
-82
binding to –35 and –10 sequences

-35

-10

-82 +11

lac
repressor
Catabolite Repression of the lac
Operon
• When glucose is present, lac operon is in
a relatively inactive state
• Selection in favor of glucose attributed to
role of a breakdown product, catabolite
• Process known as catabolite repression
uses a breakdown product to repress the
operon

7-37
 Positive Control of lac Operon
• Positive control of the
lac operon by a
substance sensing
lack of glucose that
responds by activating
the lac promoter
– The concentration of a
nucleotide, cyclic-AMP
(cAMP), rises as the
concentration of
glucose drops
Glucose 농도 감지: glucose 존재시 감소, glucose 없을 때 증가

7-38
 Catabolite Activator Protein
• cAMP added to E. coli can overcome
catabolite repression of lac operon
• Addition of cAMP lead to activation of the lac
gene even in the presence of glucose
• Positive controller of lac operon has 2 parts:
– cAMP
– Protein factor is known as:
• Catabolite activator protein or CAP or
• Cyclic-AMP receptor protein or CRP
• Gene encoding this protein is crp 7-39
 Stimulation of lac Operon
CAP-cAMP complex Stimulation of β–galactosidase synthesis by cAMP

positively controls the

activity of b-galactosidase
– CAP binds cAMP tightly 다른 side effect
– Mutant CAP not bind
cAMP tightly
– Compare activity and
production of b-
galactosidase using both
complexes
– Low activity with mutant
CAP-cAMP
Reduced affinity for cAMP 7-40
 The Mechanism of CAP Action
• CAP-cAMP complex binds to the lac promoter
– Mutants whose lac gene is not stimulated by complex
had the mutation in the lac promoter
– Mapping the DNA has shown that the activator-
binding site lies just upstream of the promoter
• Binding of CAP and cAMP to the activator site
helps RNA polymerase form an open promoter
complex

7-41
Fig. 7.16

CAP+cAMP complex allows formation of an open

promoter complex

Closed complex에만 작용

Closed complex
Rif sensitive

Open complex Rif resistant

Rif이 nucleoside보다 늦게 polymerase에 결합
 CAP
• Binding sites for CAP in lac, gal and ara operons all
contain the sequence TGTGA
– Sequence conservation suggests an important role in CAP
binding
– Binding of CAP-cAMP complex to G’s in DNA is tight
• CAP-cAMP activated operons have very weak promoters
– Their -35 boxes are quite unlike the consensus sequence
– If these promoters were strong they could be activated even
when glucose is present

Up + core

7-43
 Recruitment

• CAP-cAMP recruits polymerase to the

promoter in two steps:
– Formation of the closed promoter complex
– Conversion of the closed promoter complex
into the open promoter complex

R + P ⎯→
KB
RPc ⎯⎯→
k2
RPo
• CAP-cAMP bends its target DNA by about
100° when it binds
7-44
Fig. 7.18
Crystal structure of CAP-cAMP-αCTD-DNA
complex
80˚

100˚ Major
DNA 꺽이는 부분
(minor groove)

AT-rich which RNA polymerase

attracts αCTD

CAP-cAMP-αCTD-DNA complex CAP-cAMP-DNA complex

- αCTD 존재 유무에 상관없이 DNA가 굽어짐

- 이러한 구조에 αCTD는 영향을 주지 않음
 CAP-cAMP Promoter Complexes

Protein binding
site RE

bent

Mobility
증가

linear

7-46
Electrophoresis of CAP-cAMP-promoter complex
Proposed CAP-cAMP Activation
of lac Transcription
• The CAP-cAMP dimer
binds to its target site
on the DNA
• The aCTD (a-carboxy
terminal domain) of
polymerase interacts
with a specific site on
CAP 2개의 αCTD 중에서 한 개 만이 CAP와 결합 (ARI)

• Binding is strengthened
between promoter and Activation region I (ARI)

polymerase

7-47
 Summary

• CAP-cAMP binding to the lac activator-

binding site recruits RNA polymerase to the
adjacent lac promoter to form a closed
complex
• CAP-cAMP causes recruitment through
protein-protein interaction with the aCTD of
RNA polymerase

7-48
 7.2 The ara Operon
• The ara operon of E. coli codes for
enzymes required to metabolize the
sugar arabinose
• It is another catabolite-repressible
operon

7-49
 Control of the ara Operon

BAD

Control protein gene

Negative control
Repression loop Ara C

PBAD
Looping out the DNA and repressing the operon

Positive control Derepressed

PBAD
7-50
Arabinose
If glucose is absent, CAP-cAMP complex is in high enough concentration to
occupy the CAP binding site, which stimulate polymerase binding to the promoter.
 Features of the ara Operon

• Two ara operators exist:

– araO1 regulates transcription of a control gene
called araC
– araO2 is located far upstream of the promoter it
controls (PBAD)
• The CAP-binding site is 200 bp upstream of
the ara promoter, yet CAP stimulates
transcription
• This operon has another system of negative
regulation mediated by the AraC protein
7-51
 The ara Operon Repression Loop
• The araO2 operator controls transcription from a
promoter 250 downstream
• Does the DNA between the operator and the
promoter loop out?

7-52
 The ara Control Protein
• The AraC, ara control protein, acts as both a
positive and negative regulator
• There are 3 binding sites
• Far upstream site, araO2
• araO1 located between -106 and -144
• araI is really 2 half-sites
– araI1 between -56 and -78
– araI2 -35 to -51
– Each half-site can bind one monomer of AraC

7-53
 The araCBAD Operon

The ara operon is also called the araCBAD

operon for its 4 genes
– Three genes, araB, A, and D, encode the
arabinose metabolizing enzymes
– These are transcribed rightward from the
promoter araPBAD
– Other gene, araC
• Encodes the control protein AraC
• Transcribed leftward from the araPc promoter

7-54
 AraC Control of the ara Operon
• In absence of arabinose, no araBAD products
are needed, AraC exerts negative control
– Binds to araO2 and araI1
– Loops out the DNA in between
– Represses the operon
• Presence of arabinose, AraC changes
conformation
– It can no longer bind to araO2
– Occupies araI1 and araI2 instead
– Repression loop broken
– Operon is derepressed

7-55
 Control of the ara Operon

BAD

Control protein gene

Negative control
Repression loop Ara C

PBAD
Looping out the DNA and repressing the operon

Positive control Derepressed

PBAD
7-56
Arabinose
If glucose is absent, CAP-cAMP complex is in high enough concentration to
occupy the CAP binding site, which stimulate polymerase binding to the promoter.
 Positive Control of the ara Operon
• Positive control is also mediated by CAP
and cAMP
• The CAP-cAMP complex attaches to its
binding site upstream of the araBAD
promoter
• DNA looping would allow CAP to contact
the polymerase and thereby stimulate its
binding to the promoter

7-57
Fig. 7.23
Fig. 7.24
Fig. 7.25
Fig. 7.26
Fig. 7.27
 ara Operon Summary
• The ara operon is controlled by the AraC protein
– Represses by looping out the DNA between 2 sites,
araO2 and araI1 that are 210 bp apart
• Arabinose can derepress the operon causing
AraC to loosen its attachment to araO2 and bind
to araI2
– This breaks the loop and allows transcription of operon
• CAP and cAMP stimulate transcription by binding
to a site upstream of araI
– AraC controls its own synthesis by binding to araO1
and prevents leftward transcription of the araC gene
7-63
 7.3 The trp Operon
• The E. coli trp operon contains the genes for the
enzymes the bacterium needs to make the
amino acid tryptophan
• The trp operon codes for anabolic enzymes,
those that build up a substance
• Anabolic enzymes are typically turned off by a
high level of the substance produced
• This operon is subject to negative control by a
repressor when tryptophan levels are elevated
• trp operon also exhibits attenuation

7-64
65
Tryptophan’s Role in Negative Control of
the trp Operon
• Five genes code for the polypeptides in
the enzymes of tryptophan synthesis
• The trp operator lies wholly within the trp
promoter
• High tryptophan concentration is the signal
to turn off the operon
• The presence of tryptophan helps the trp
repressor bind to its operator
7-66
 Negative Control of the trp Operon
Derepression
1) Standard negative control
• Without tryptophan no trp
repressor exists, just the
inactive protein,
aporepressor
• If aporepressor binds
tryptophan, changes
Negative control =
conformation with high Repression
affinity for trp operator
• Combine aporepressor and
tryptophan to form the trp
repressor
• Tryptophan is a
corepressor F7.25
Corepressor 7-67
Repression =70X
Attenuation =10X: repression 기작이 약해서 repressor가 있는 조건에서도 transcription이 여전히 일어남

2) Attenuation in the trp Operon F7.25

ORF

Two hairpin 구조(transcription stop signal)

ORF

7-68
F7.28
T155
 Mechanism of Attenuation
• Attenuation imposes an extra
level of control on an operon,
more than just the repressor-
operator system (repression 70x ;
attenuation 10x)
• Operates by causing premature
termination of the operon’s
transcript when product is
abundant
• In the presence of low tryptophan
concentration, the RNA
polymerase reads through the
attenuator so the structural genes
are transcribed

• In the presence of high tryptophan

concentration, the attenuator
Terminator hairpin
causes premature termination of
7-69
transcription
Fig. 7.27
Two sturctures available to the leader-attenuator transcript
1) transcription/translation이 같은 속도로 일어남
2) Trp codon이 두개 연속하여 위치
3) ¾ hair pin 구조가 ρ(rho)-dependent terminator 구조와 유사 (F6.41)

More stable Less stable

Trp

Trp
Leader Leader

U8

Attenuator 2/3

Ribosome

1/2
Trp Trp
No Attenuator

High Trp 3/4 Similar to rho(ρ) independent terminator

Low Trp
 Defeating (Overriding) Attenuation
• Attenuation operates in the E. coli trp
operon as long as tryptophan is plentiful
• If amino acid supply low, ribosomes stall at
the tandem tryptophan codons in the trp
leader
• trp leader being synthesized as stalling
occurs, stalled ribosome will influence the
way RNA folds
– Prevents formation of a hairpin (terminator)
– This is part of the transcription termination
signal which causes attenuation
7-71
Fig. 7.29 7.27과는 좌우 반대로 그려져 있음

Overriding Attenuation

U8

Attenuator

Low Trp High Trp

Q. 왼쪽(Low Trp)그림에서 Ribosome이 Trp codon에 머물러 있으면 TrpE 유전자는 어떻게 translation
될까?
7-72
Watson et al. 1987 Molecular Biology of the Gene 4th ed.

73
 7.4 Riboswitches

• Small molecules can act directly on the 5’-

UTRs of mRNAs to control their
expression
• Regions of 5’-UTRs capable of altering
their structures to control gene expression
in response to ligand binding are called
riboswitches

7-74
 Riboswitch Action
• Region that binds to the ligand is an
aptamer
• An expression platform is another module
in the riboswitch which can be:
– Terminator
– Ribosome-binding site
– Another RNA element that affects gene
expression
• Operates by depressing gene expression
– Some work at the transcriptional level
– Others can function at the translational level
7-75
(Chapter 17)
Fig. 7.34

Results of in-line probing of RFN element and mode for the action of ribD riboswitch

RFN element
riboflavin
Spontaneous cleavage Less susceptible to cleavage Flavin mononucleotide
FMN

(-)FMN (+)FMN

ribD operon (riboflavin 합성)의 product

(-)FMN: Unstructured –spontaneous cleavage (more susceptible to cleavage)

(+)FMN: 2˚ structure by intramolecular base pairing or by binding to a ligand –
less susceptible to cleavage
 Model of Riboswitch Action
• FMN binds to aptamer (RFN element)
in a riboswitch called the
RFN element in 5’-UTR
of the ribD mRNA
• Binding FMN, base
pairing in riboswitch Terminator
of RBS
changes to create a (FMN)

terminator
• Transcription is
attenuated
• Saves cell energy as
FMN is a product of the Allosteric
ribD operon effect
7-77
TABLES
Tab. 7.1
Tab. 7.2
Page 200
Page 200