Transcript Gene7-10

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 cis-acting
sites in order for RNA polymerase to initiate transcription at the promoter.
In a eukaryotic system, a structural gene is controlled individually.
10.2 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 cisacting, 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
poor-growth 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 ribosome-binding
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 140-base 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.