Chapter
10
The operon
10.1 Introduction
10.2 Regulation can be negative or positive
10.3 Structural gene clusters are coordinately controlled
10.4 The lac genes are controlled by a repressor
10.5 The lac operon can be induced
10.6 Repressor is controlled by a small molecule inducer
10.7 cis-acting constitutive mutations identify the operator
10.8 trans-acting mutations identify the regulator gene
10.9 Multimeric proteins have special genetic properties
10.10 Repressor protein binds to the operator
10.11 Binding of inducer releases repressor from the operator
10.12 Repressor is a tetramer
10.13 Repressor binds to three operators and interacts with RNA
polymerase
10.14 Repressor is always bound to DNA
10.15 The operator competes with low-affinity sites to bind repressor
10.16 Repression can occur at multiple loci
10.17 Distinguishing positive and negative control
10.18 Catabolite repression involves the inducer cyclic AMP and the activator
CAP
10.19 CAP functions in different ways in different target operons
10.20 CAP bends DNA
10.21 The stringent response produces (p)ppGpp
10.22 (p)ppGpp is produced by the ribosome
10.23 pGpp has many effects
10.24 Translation can be regulated
10.25 r-protein synthesis is controlled by autogeneous regulation
10.26 Phage T4 p32 is controlled by an autogenous circuit
10.27 Autogenous regulation is often used to control synthesis of
macromolecular assemblies
10.28 Alternative secondary structures control attenuation
10.29 The tryptophan operon is controlled by attenuation
10.30 Attenuation can be controlled by translation
10.31 Small RNA molecules can regulate translation
10.32 Antisense RNA can be used to inactivate gene expression
10.1 Introduction
Operator is the site on DNA at which a
repressor protein binds to prevent
transcription from initiating at the
adjacent promoter.
Repressor protein binds to operator on
DNA or RNA to prevent transcription or
translation, respectively.
Structural gene codes for any RNA or
protein product other than a regulator.
10.1 Introduction
Figure 10.1 A
regulator gene
codes for a
protein that acts
at a target site
on DNA.
10.1 Introduction
Figure 10.2 In
negative control,
a trans-acting
repressor binds
to the cis-acting
operator to turn
off
transcription. In
prokaryotes,
multiple genes
are controlled
coordinately.
10.1 Introduction
Figure 10.3 In positive control, trans-acting factors must bind to cisacting sites in order for RNA polymerase to initiate transcription at the
promoter. In a eukaryotic system, a structural gene is controlled
individually.
10.2 Structural gene clusters are
coordinately controlled
Operon is a unit of bacterial gene
expression and regulation, including
structural genes and control elements in
DNA recognized by regulator gene
product(s).
10.2 Structural gene clusters are coordinately controlled
Figure 10.4 The lac operon occupies ~6000 bp of DNA. At the
left the lacI gene has its own promoter and terminator. The
end of the lacI region is adjacent to the promoter, P. The
operator, O, occupies the first 26 bp of the long lacZ gene,
followed by the lacY and lacA genes and a terminator.
10.2 Structural gene clusters are coordinately controlled
Figure 10.5
Repressor and
RNA
polymerase bind
at sites that
overlap around
the startpoint of
the lac operon.
10.3 Repressor is controlled by a small molecule inducer
Allosteric control refers to the ability of an interaction at one site of a protein
to influence the activity of another site.
Coordinate regulation refers to the common control of a group of genes.
Corepressor is a small molecule that triggers repression of transcription by
binding to a regulator protein.
Gratuitous inducers resemble authentic inducers of transcription but are not
substrates for the induced enzymes.
Inducer is a small molecule that triggers gene transcription by binding to a
regulator protein.
Induction refers to the ability of bacteria (or yeast) to synthesize certain
enzymes only when their substrates are present; applied to gene expression,
refers to switching on transcription as a result of interaction of the inducer
with the regulator protein.
Repression is the ability of bacteria to prevent synthesis of certain enzymes
when their products are present; more generally, refers to inhibition of
transcription (or translation) by binding of repressor protein to a specific site
on DNA (or mRNA).
10.3 Repressor is controlled by a small molecule inducer
Figure 10.6 Addition of
inducer results in rapid
induction of lac mRNA,
and is followed after a
short lag by synthesis of
the enzymes; removal of
inducer is followed by
rapid cessation of
synthesis.
10.3 Repressor is controlled by a small molecule inducer
Figure 10.7 Repressor
maintains the lac
operon in the inactive
condition by binding
to the operator;
addition of inducer
releases the repressor,
and thereby allows
RNA polymerase to
initiate transcription.
10.4 Mutations identify the operator and the regulator gene
Interallelic complementation describes the change
in the properties of a heteromultimeric protein
brought about by the interaction of subunits coded
by two different mutant alleles; the mixed protein
may be more or less active than the protein
consisting of subunits only of one or the other
type.
Negative complementation occurs when
interallelic complementation allows a mutant
subunit to suppress the activity of a wild-type
subunit in a multimeric protein.
10.4 Mutations identify the operator and the regulator gene
Figure 10.8
Operator mutations
are constitutive
because the operator
is unable to bind
repressor protein;
this allows RNA
polymerase to have
unrestrained access
to the promoter. The
Oc mutations are
cis-acting, because
they affect only the
contiguous set of
structural genes.
10.4 Mutations identify the operator and the regulator gene
Figure 10.9
Mutations that
inactivate the
lacI gene cause
the operon to be
constitutively
expressed,
because the
mutant repressor
protein cannot
bind to the
operator.
10.4 Mutations identify the operator and the regulator gene
Figure 10.10 Mutations map the regions of the lacl gene responsible for different
functions. The DNA-binding domain is identified by lacI-d mutations at the Nterminal region; lacl- mutations unable to form tetramers are located between
residues 220-280. Other lacI- mutations occur throughout the gene. lacIs
mutations occur in regularly spaced clusters between residues 62-300.
10.5 Repressor protein binds to the operator and is released by inducer
Figure 10.11
The lac operator
has a
symmetrical
sequence. The
sequence is
numbered
relative to the
startpoint for
transcription at
+1. The regions
of dyad
symmetry are
indicated by the
shaded blocks.
10.5 Repressor protein binds to the operator and is released by inducer
Figure 9.16 One face of the promoter contains
the contact points for RNA.
10.5 Repressor protein binds to
the operator and is released by
inducer
Figure 10.12
Does the
inducer bind to
free repressor to
upset an
equilibrium (left)
or directly to
repressor bound
at the operator
(right)?
10.5 Repressor protein binds to
the operator and is released by
inducer
Figure 10.13 The
structure of a
monomer of Lac
repressor identifies
several independent
domains.
Photograph kindly
provided by
Mitchell Lewis.
10.5 Repressor protein binds to
the operator and is released by
inducer
Figure 10.14
The crystal structure of
the core region of Lac
repressor identifies the
interactions between
monomers in the
tetramer. Each monomer
is identified by a different
color. Photographs kindly
provided by Alan
Friedman.
10.5 Repressor protein binds to
the operator and is released by
inducer
Figure 10.14
The crystal structure of
the core region of Lac
repressor identifies the
interactions between
monomers in the
tetramer. Each
monomer is identified
by a different color.
Photographs kindly
provided by Alan
Friedman.
10.5 Repressor protein binds to
the operator and is released by
inducer
Figure 10.14
The crystal structure of
the core region of Lac
repressor identifies the
interactions between
monomers in the
tetramer. Each monomer
is identified by a different
color. Photographs kindly
provided by Alan
Friedman.
10.5 Repressor protein binds to
the operator and is released by
inducer
Figure 10.14
The crystal structure of
the core region of Lac
repressor identifies the
interactions between
monomers in the
tetramer. Each monomer
is identified by a different
color. Photographs kindly
provided by Alan
Friedman.
10.5 Repressor protein binds to
the operator and is released by
inducer
Figure 10.15 Inducer
changes the structure of
the core so that the
headpieces of a repressor
dimer are no longer in an
orientation that permits
binding to DNA.
Photographs kindly
provided by Mitchell
Lewis.
10.5 Repressor protein binds to
the operator and is released by
inducer
Figure 10.16 When a
repressor tetramer binds to
two operators, the stretch
of DNA between them is
forced into a tight loop.
(The blue structure in the
center of the looped DNA
represents CAP, another
regulator protein that binds
in this region). Photograph
kindly provided by Mitchell
Lewis.
10.6 The specificity of protein-DNA interactions
Figure 10.17 Lac repressor binds strongly and specifically
to its operator, but is released by inducer. All equilibrium
constants are in M-1.
10.6 The specificity of protein-DNA interactions
Figure 10.18
Virtually all the
repressor in the
cell is bound to
DNA.
10.6 The specificity
of protein-DNA
interactions
Figure 9.12
How does RNA
polymerase find
target promoters
so rapidly on
DNA?
10.7 Repression can occur at multiple loci
Autogenous control describes the action of a
gene product that either inhibits (negative
autogenous control) or activates (positive
autogenous control) expression of the gene
coding for it.
10.7 Repression can occur at multiple loci
Figure 10.19 The trp repressor recognizes operators at three loci.
Conserved bases are shown in red. The location of the mRNA varies,
as indicated by the red arrows.
10.7 Repression can
occur at multiple
loci
Figure 10.20
Operators may lie
at various
positions relative
to the promoter.
10.8 Distinguishing positive and negative
control
Derepressed state describes a gene that is turned
on. It is synonymous with induced when
describing the normal state of a gene; it has the
same meaning as constitutive in describing the
effect of mutation.
10.8 Distinguishing
positive and
negative control
Figure 10.2
In negative control, a
trans-acting
repressor binds to
the cis-acting
operator to turn off
transcription. In
prokaryotes,
multiple genes are
controlled
coordinately.
10.8 Distinguishing positive and negative control
Figure 10.3 In positive control, trans-acting factors must bind to cis-acting sites in
order for RNA polymerase to initiate transcription at the promoter. In a
eukaryotic system, a structural gene is controlled individually.
10.8
Distinguishing
positive and
negative control
Figure 10.21
Control circuits
are versatile and
can be designed to
allow positive or
negative control
of induction or
repression.
10.9 Catabolite repression involves positive
regulation at the promoter
Catabolite repression describes the decreased
expression of many bacterial operons that results
from addition of glucose. It is caused by a decrease
in the level of cyclic AMP, which in turn inactivates
the CAP regulator.
10.9 Catabolite repression involves positive
regulation at the promoter
Figure 10.22 Cyclic AMP has a single phosphate group
connected to both the 3￠ and 5￠ positions of the sugar ring.
10.9 Catabolite
repression involves
positive regulation
at the promoter
Figure 10.21
Control circuits
are versatile and
can be designed to
allow positive or
negative control
of induction or
repression.
10.9 Catabolite
repression involves
positive regulation
at the promoter
Figure 10.23
Glucose causes
catabolite
repression by
reducing the
level of cyclic
AMP.
10.9 Catabolite repression involves
positive regulation at the promoter
Figure 10.24 The
consensus
sequence for CAP
contains the well
conserved
pentamer TGTGA
and (sometimes)
an inversion of
this sequence
(TCANA).
10.9 Catabolite repression involves positive
regulation at the promoter
Figure 10.25
The CAP
protein can
bind at
different sites
relative to
RNA
polymerase.
10.9 Catabolite repression involves positive
regulation at the promoter
Figure 10.26 Gel electrophoresis can be used to analyze
bending.
10.9 Catabolite repression involves positive
regulation at the promoter
Figure 10.27
CAP bends DNA
>90° around the
center of
symmetry.
10.10 Adverse growth conditions provoke the
stringent response
Idling reaction is the production of pppGpp and
ppGpp by ribosomes when an uncharged tRNA is
present in the A site; triggers the stringent response.
Stringent response refers to the ability of a bacterium to
shut down synthesis of tRNA and ribosomes in a poorgrowth medium.
10.10 Adverse growth conditions provoke the
stringent response
Figure 10.28
Stringent factor
catalyzes the
synthesis of
pppGpp and
ppGpp; ribosomal
proteins can
dephosphorylate
pppGpp to ppGpp.
10.10 Adverse growth conditions provoke the
stringent response
Figure 10.29 In normal
protein synthesis, the
presence of aminoacyl-tRNA
in the A site is a signal for
peptidyl transferase to
transfer the polypeptide
chain, followed by movement
catalyzed by EF-G; but
under stringent conditions,
the presence of uncharged
tRNA causes RelA protein to
synthesize (p)ppGpp and to
expel the tRNA.
10.10 Adverse
growth conditions
provoke the
stringent response
Figure 10.30
Nucleotide levels
control initiation
of rRNA
transcription.
10.10 Adverse
growth conditions
provoke the
stringent response
Figure 10.35
Translation of the rprotein operons is
autogenously
controlled and
responds to the level
of rRNA.
10.11 Autogenous control may occur at
translation
Figure 10.31 A
regulator protein
may block
translation by
binding to a site on
mRNA that overlaps
the ribosomebinding site at the
initiation codon.
10.11 Autogenous control may occur at
translation
Figure 10.32 Proteins that bind to sequences
within the initiation regions of mRNAs may
function as translational repressors.
10.11 Autogenous
control may occur
at translation
Figure 10.33 Secondary
structure can control
initiation. Only one
initiation site is available in
the RNA phage, but
translation of the first
cistron changes the
conformation of the RNA
so that other initiation site(s)
become available.
10.11 Autogenous
control may occur
at translation
Figure 10.34 Genes for
ribosomal proteins,
protein synthesis factors,
and RNA polymerase
subunits are interspersed
in a small number of
operons that are
autonomously regulated.
The regulator is named in
red; the proteins that are
regulated are shaded in
pink.
10.11 Autogenous
control may occur
at translation
Figure 10.35
Translation of the rprotein operons is
autogenously
controlled and
responds to the level
of rRNA.
10.11 Autogenous
control may occur
at translation
Figure 10.36 Excess
gene 32 protein (p32)
binds to its own
mRNA to prevent
ribosomes from
initiating translation.
10.11 Autogenous control may occur at
translation
Figure 10.37 Gene 32
protein binds to various
substrates with different
affinities, in the order
single-stranded DNA,
its own mRNA, and
other mRNAs. Binding
to its own mRNA
prevents the level of p32
from rising >10-6 M.
10.11 Autogenous
control may occur
at translation
Figure 10.38 Tubulin is
assembled into microtubules
when it is synthesized.
Accumulation of excess free
tubulin induces instability in
the tubulin mRNA by acting
at a site at the start of the
reading frame in mRNA or at
the corresponding position in
the nascent protein.
10.12 Alternative
secondary structures
control attenuation
Figure 10.39
Attenuation
occurs when a
terminator hairpin
in RNA is
prevented from
forming.
10.13 Attenuation
can be controlled by
translation
Figure 10.40
Termination can
be controlled via
changes in RNA
secondary
structure that are
determined by
ribosome
movement.
10.13 Attenuation can be controlled by translation
Figure 10.41
The trp operon
consists of five
contiguous
structural
genes preceded
by a control
region that
includes a
promoter,
operator, leader
peptide coding
region, and
attenuator.
10.13 Attenuation
can be controlled
by translation
Figure 10.42 An attenuator
controls the progression of
RNA polymerase into the trp
genes. RNA polymerase
initiates at the promoter and
then proceeds to position 90,
where it pauses before
proceeding to the attenuator at
position 140. In the absence of
tryptophan, the polymerase
continues into the structural
genes (trpE starts at +163). In
the presence of tryptophan
there is ~90% probability of
termination to release the 140base leader RNA.
10.13 Attenuation can be controlled by translation
Figure 10.43 The trp leader region can exist in alternative base-paired conformations. The center
shows the four regions that can base pair. Region 1 is complementary to region 2, which is
complementary to region 3, which is complementary to region 4. On the left is the conformation
produced when region 1 pairs with region 2, and region 3 pairs with region 4. On the right is the
conformation when region 2 pairs with region 3, leaving regions 1 and 4 unpaired.
10.13 Attenuation
can be controlled
by translation
Figure 10.44
The alternatives for
RNA polymerase at
the attenuator
depend on the
location of the
ribosome, which
determines whether
regions 3 and 4 can
pair to form the
terminator hairpin.
10.14 Small RNA
molecules can
regulate translation
Figure 10.45
Antisense RNA
can affect
function or
stability of an
RNA target.
10.14 Small RNA
molecules can
regulate translation
Figure 10.46 Increase in
osmolarity activates EnvZ,
which activates OmpR,
which induces
transcription of micF and
ompC (not shown). micF
RNA is complementary to
the 5￠ region of ompF
mRNA and prevents its
translation.
10.14 Small RNA
molecules can
regulate translation
Figure 10.47 lin4
RNA regulates
expression of
lin14 by binding
to the 3￠
nontranslated
region.
10.14 Small RNA molecules can regulate translation
Figure 10.48
Antisense
RNA can be
generated by
reversing the
orientation of
a gene with
respect to its
promoter, and
can anneal
with the wildtype transcript
to form
duplex RNA.
Summary
1. Transcription is regulated by the interaction between
trans-acting factors and cis-acting sites.
2. Initiation of transcription is regulated by interactions
that occur in the vicinity of the promoter.
3. A repressor protein prevents RNA polymerase either
from binding to the promoter or from activating
transcription.
4. The ability of the repressor protein to bind to its
operator is regulated by a small molecule.
Summary
5. The lactose pathway operates by induction, when an inducer galactoside prevents the repressor from binding its operator;
transcription and translation of the lacZ gene then produce galactosidase, the enzyme that metabolizes -galactosides.
6. Some promoters cannot be recognized by RNA polymerase (or are
recognized only poorly) unless a specific activator protein is present.
7. A protein with a high affinity for a particular target sequence in
DNA has a lower affinity for all DNA.
8. Gene expression can be controlled at stages subsequent to
transcription.
9. The level of protein synthesis itself provides an important
coordinating signal.