Liam.Keegan@ceitec.muni.cz
Lewin, GENES XI
Chapter 26
Operons, prokaryotic gene regulation and
yeast GAL4
Lewin’s GENES XII (PDF)
https://pdfroom.com/books/lewins-genes-xii/Wx5aDYKl2BJ
biotecher.ir
https://biotecher.ir/wp-content/uploads/2018/06/bio...
Chapter 26
The Operon
26.2 Operons are Structural Gene Clusters
that Are Coordinately Controlled
• Genes coding for proteins that function in the same
pathway may be located adjacent to one another and
controlled as a single unit that is transcribed into a
polycistronic mRNA.
Figure 26.05: The lac operon occupies ~6000 bp of DNA.
Sequence-specific DNA recognition is the key to differential
gene regulation.
Mark Ptashne with graduate students Cynthia Wohlberger, Liam Keegan, Ed Giniger at Harvard, 1982
26.1 Introduction
• In negative regulation, a repressor protein binds to an
operator to prevent a gene from being expressed.
• In positive regulation, a transcription factor is required
to bind at the promoter in order to enable RNA
polymerase to initiate transcription.
Figure 26.02: In negative control, a transacting
repressor binds to the cis-acting
operator to turn off transcription.
Figure 26.03: In positive control, a transacting
factor must bind to cis-acting site in
order for RNA polymerase to initiate
transcription at the promoter.
26.1 Introduction
• In inducible regulation, the gene is regulated by the
presence of its substrate (the inducer).
• In repressible regulation, the gene is regulated by the
product of its enzyme pathway (the corepressor).
26.3 Famous inducible genes in E. coli
The lac Operon Is under Negative control by a repressor
• Transcription of the lacZYA
operon is controlled by a
repressor protein (the lac
repressor) that binds to an
operator that overlaps the
promoter at the start of the
cluster.
• constitutive expression –
A state in which a gene is
expressed continuously.
• In the absence of βgalactosides,
the lac operon
is expressed only at a very
low (basal) level.
Figure 26.06: lac repressor and RNA
polymerase bind at sites that overlap
around the transcription startpoint of the
lac operon.
ONPG hydrolysis by cells gives a soluble blue product for
spectrophotometric measurement of lacZ (β-galactosidase ) activity units
lacZ (β-galactosidase)) plate tests and liquid culture assays for
beta-gal activity units.
26.3 The lac Operon Is under negative
transcriptional control by repressor and is
inducible by galactosides
• The repressor protein is a tetramer of identical subunits
coded by the lacI gene.
• β-galactoside sugars, the substrates of the lac operon,
are its inducer.
• Addition of specific β-galactosides induces transcription
of all three genes of the lac operon.
• The lac mRNA is extremely unstable; as a result,
induction can be rapidly reversed.
26.3 The lac Operon Is Negative Inducible
Figure 26.07: Addition of inducer results in rapid induction of lac mRNA, and is followed
after a short lag by synthesis of the enzymes.
26.4 lac Repressor Is Controlled by a
Small-Molecule Inducer
• An inducer functions by
converting the repressor
protein into a form with lower
operator affinity.
• Repressor has two binding
sites, one for the operator DNA
and another for the inducer.
• gratuitous inducer – Inducers
that resemble authentic
inducers of transcription, but
are not substrates for the
induced enzymes.Figure 26.08: lac repressor maintains the
lac operon in the inactive condition by
binding to the operator.
26.4 lac Repressor Is Controlled by a
Small-Molecule Inducer
• Repressor is inactivated by an allosteric interaction in which binding of
inducer at its site changes the properties of the DNA-binding site (allosteric
control).
• The true inducer is allolactose, not the actual substrate of β-galactosidase.
In experiments we induce with artificial IPTG, not metabolized, a gratuitous
inducer.
Figure 26.09: Addition of inducer converts
repressor to a form with low affinity for the
operator. This allows RNA polymerase to
initiate transcription.
26.5 cis-Acting Constitutive Mutations
Identify the Operator
• Mutations in the operator cause constitutive expression
of all three lac structural genes.
• These mutations are cis-acting and affect only those
genes on the contiguous stretch of DNA.
• Mutations in the promoter prevent expression of lacZYA
and are uninducible and cis-acting.
26.5 cis-Acting Constitutive Mutations
Identify the Operator
• cis-dominant – A site or mutation that affects the properties only of
its own molecule of DNA, often indicating that a site does not code
for a diffusible product. OC means ‘operator constitutive’ mutation
Figure 26.10: Operator mutations are
constitutive because the operator is
unable to bind repressor protein.
26.6 trans-Acting Mutations Identify the
Regulator Gene
• Mutations in the lacI gene are trans-acting and affect
expression of all lacZYA clusters in the bacterium.
• Mutations that eliminate lacI function cause constitutive
expression and are recessive (lacI–).
• Mutations in the DNA-binding
site of the repressor are
constitutive because the
repressor cannot bind the
operator.
Figure 26.11: Mutations that inactivate the
lacI gene cause the operon to be
constitutively expressed.
26.6 trans-Acting Mutations Identify the
Regulator Gene
• Mutations in the inducer-binding site of the
repressor prevent it from being inactivated and
cause uninducibility. Super-repressor mutants.
• When mutant and wild-type subunits are
present, a single lacI–d mutant subunit can
inactivate a tetramer whose other subunits are
wild-type.
– It is dominant negative.
26.6 trans-Acting Mutations Identify the
Regulator Gene
• negative complementation
– This occurs when interallelic
complementation allows a
mutant subunit to suppress
the activity of a wild-type
subunit in a multimeric
protein.
• lacI–d mutations occur in the
DNA-binding domain. Their
effect is explained by the fact
that repressor activity
requires all DNA-binding sites
in the tetramer to be active.
Figure 26.12: A lacI-d mutant gene makes a
monomer that has a damaged DNA binding.
26.7 lac Repressor Is a Tetramer Made of
Two Dimers
• A single repressor subunit can be divided into the Nterminal
DNA-binding domain, a hinge, and the core of
the protein.
• The DNA-binding domain contains two short α-helical
regions that bind the major groove of DNA.
• The inducer-binding site and the regions responsible for
multimerization are located in the core.
26.7 lac Repressor Is a Tetramer Made of
Two Dimers
Figure 26.13: The structure of a monomer of Lac repressor identifies several independent
domains.
Structure from Protein Data Bank 1LBG. M. Lewis, et al.,
Science 271 (1996): 1247-1254. Photo courtesy of Hongli
Zhan and Kathleen S. Matthews, Rice University.
26.7 lac Repressor Is a Tetramer Made of
Two Dimers
• Monomers form a dimer by
making contacts between
core subdomains 1 and 2.
• Dimers form a tetramer by
interactions between the
tetramerization helices.
Figure 26.15: The repressor tetramer
consists of two dimers.
26.7 lac Repressor Is a Tetramer Made of
Two Dimers
• Different types of mutations
occur in different domains
of the repressor protein.
Figure 26.16: The locations of three type of
mutations in lactose repressor are mapped on
the domain structure of the protein.
26.8 lac Repressor Binding to the Operator
Is Regulated by an Allosteric Change in
Conformation
• Inducer binding causes a
change in repressor
conformation that reduces its
affinity for DNA and releases
it from the operator.
Figure 26.18: The inducer changes the
structure of the core.
26.8 lac Repressor Binding to the Operator
Is Regulated by an Allosteric Change in
Conformation
• lac repressor protein binds to the double-stranded DNA
sequence of the operator.
• The operator is a palindromic sequence of 26 bp.
• Each inverted repeat of the operator binds to the DNAbinding
site of one repressor subunit.
Figure 26.17: The lac operator has a symmetrical sequence.
26.9 lac Repressor Binds to Three
Operators and Interacts with RNA
Polymerase
• Each dimer in a repressor
tetramer can bind an operator, so
that the tetramer can bind two
operators simultaneously.
• Full repression requires the
repressor to bind to an additional
operator downstream or upstream
as well as to the primary operator
at the lacZ promoter.
• Binding of repressor at the
operator stimulates binding of
RNA polymerase at the promoter
but precludes transcription.
Figure 26.21: If both dimers in a
repressor tetramer bind to DNA,
the DNA between the two binding
sites is held in a loop. Third
operator not shown.
26.11 Positive regulators of transcription
The lac Operon Has a Second Layer of Control:
Catabolite Repression
• catabolite repression – The ability of glucose
to prevent the expression of a number of genes.
– In bacteria this is a positive control system; in
eukaryotes, it is completely different.
• Catabolite repressor protein (CRP) is an
activator protein that binds to a target sequence
at a promoter.
• CRP is or was also known as cAMP-dependent,
Catabolite Activator Protein (CAP), a newer
name I find clearer.
26.11 The lac Operon Has a Second Layer
of Control: Catabolite Repression
Figure 26.25: cAMP converts an activator protein CRP to a form that binds the promoter
and assists RNA polymerase in initiating transcription.
26.11 The lac Operon Has a Second Layer
of Control: Catabolite Repression
• A dimer of CRP is activated by
a single molecule of cyclic
AMP (cAMP).
• cAMP is controlled by the
level of glucose in the cell; a
low glucose level allows
cAMP to be made.
• CRP interacts with the Cterminal
domain of the α
subunit of RNA polymerase to
activate it.Figure 26.27: By reducing the level of
cyclic AMP, glucose inhibits the
transcription of operons that require
CRP activity.
27.1 Introduction
Famous inducible genes and
transcriptional repressors in E. coli
• bacteriophage (or phage) – A bacterial virus.
• lytic infection – Infection of a bacterium by a phage that
ends in the destruction of the bacterium with release of
progeny phage.
• lysis – The death of bacteria at the end of a phage
infective cycle when they burst open to release the
progeny of an infecting phage (because phage enzymes
disrupt the bacterium’s cytoplasmic membrane or cell
wall).
27.1 Introduction
Figure 27.01: Lytic development involves
the reproduction of phage particles with
destruction of the host bacterium.
λ bacteriophage plaques on a lawn of E. coli are clear where all cells
were killed by virus infection and cell lysis (- like a coldsore).
Cloudy plaques contain λ lysogen cells that are immune to further
lambda virus infection leading directly to cell lysis
27.9 The Lambda Repressor and Its
Operators Define the Immunity Region
• immunity – In phages, the ability of a prophage to
prevent another phage of the same type from infecting a
cell.
• virulent mutations – Phage mutants that are unable to
establish lysogeny.
27.9 The Lambda Repressor and Its
Operators Define the Immunity Region
• Several lambdoid phages have different immunity
regions.
• A lysogenic phage confers immunity to further infection
by any other phage with the same immunity region.
Figure 27.16: In the absence
of repressor, RNA polymerase
initiates at the left and right
promoters.
27.8 Lysogeny Is Maintained by the
Lambda Repressor Protein
• The lambda repressor,
encoded by the cI gene, is
required to maintain lysogeny.
• The lambda repressor acts at
the OL and OR operators to
block transcription of the
immediate early genes.
• The immediate early genes
trigger a regulatory cascade;
as a result, their repression
prevents the lytic cycle from
proceeding.
Figure 27.15: Repressor acts at the
left operator and right operator to
prevent transcription of the
immediate early genes (N and cro).
Need to purify scarce gene regulator proteins drove
biotechnology of protein overexpression
• Take E. coli cell as a cube 1 micron on each side. A liter is a
cube 10 cm on each side. What is the concentration of the lac
operator? Just how scarce can repressor be in a cell? 10-100
molecules if the kd for operator-binding is 10-10 M or higher.
• Lambda repressor was first isolated from an overproducer
mutant virus. Later the repressor gene was expressed from lac
or tac promoters. Also, a hybrid ribosome binding site
upstream of ATG gave strong translation initiation.
27.10 The DNA-Binding Form of the
Lambda Repressor Is a Dimer
• A repressor monomer has two
distinct domains.
• The N-terminal domain contains
the DNA-binding site.
• The C-terminal domain dimerizes.
• Binding to the operator requires
the dimeric form so that two DNAbinding
domains can contact the
operator simultaneously.
• Cleavage of the repressor between
the two domains reduces the
affinity for the operator and
induces a lytic cycle.
Figure 27.18: Repressor dimers bind to the operator.
27.11 Lambda Repressor Uses a HelixTurn-Helix
Motif to Bind DNA
• Each DNA-binding region in the repressor contacts a
half-site in the DNA.
• The DNA-binding site of the repressor includes two short
α-helical regions that fit into the successive turns of the
major groove of DNA (helix-turn-helix).
• A DNA-binding site is a (partially) palindromic sequence
of 17 bp.
Figure 27.19: The operator is a 17-bp sequence with an axis of symmetry through the
central base pair.
Lambda repressor binds as a symmetrical protein dimer to a
nearly symmetrical 17 bp DNA sequence.
Symmetric repressor site 5’ T A T C A C C G C C G G T G A T A
A T A G T G G C G G C C A C T A T 5’
27.11 Lambda Repressor Uses a HelixTurn-Helix
Motif to Bind DNA
• The amino acid sequence of the recognition helix
makes contacts with particular bases in the operator
sequence that it recognizes.
Figure 27.22: Two proteins that use the
two-helix arrangement to contact DNA
recognize lambda operators with affinities
determined by helix-3.
Recognition helix amino acid side-chains make many sequencespecific
contacts to base pairs.
5’ P
3’ OH
5’ T A T C A C C G C C G G T G A T A
A T A G T G G C G G C C A C T A T 5’
Example of an amino acid contact that can discriminate
between bases in a binding site.
The position of the glutamine
is fixed when repressor binds.
Glutamine side chain needs to find the
hydrogen donor and acceptor sites in just
the right places.
It cannot reach the paired base on the
other strand.
It cannot reach the other bases on the
same strand.
Only an A base meets the criteria.
27.12 Lambda Repressor Dimers Bind
Cooperatively to the Operator
• Repressor binding to one operator increases the affinity
for binding a second repressor dimer to the adjacent
operator.
• The affinity is 10× greater for OL1 and OR1 than other
operators, so they are bound first.
• Cooperativity allows repressor to bind the OL2/OR2 sites
at lower concentrations.
Figure 27.25: When two lambda
repressor dimers bind cooperatively,
each of the subunits of one dimer
contacts a subunit in the other dimer.
27.13 Lambda Repressor Maintains an
Autoregulatory Circuit
• The DNA-binding region of repressor at OR2 contacts
RNA polymerase and stabilizes its binding to PRM.
• This is the basis for the autoregulatory control of
repressor maintenance.
• Repressor binding at OL blocks transcription of gene N
from PL.
Figure 27.26: Positive control mutations
identify a small region at helix-2 that
interacts directly with RNA polymerase.
27.13 Lambda Repressor Maintains an
Autoregulatory Circuit
• Repressor binding at OR
blocks transcription of cro,
but also is required for
transcription of cI.
• Repressor binding to the
operators therefore
simultaneously blocks entry
to the lytic cycle and
promotes its own synthesis.
Figure 27.27: Lysogeny is maintained by
an autoregulatory circuit.
27.14 Cooperative Interactions Increase the
Sensitivity of Regulation
• Repressor dimers bound at OL1 and OL2 interact with
dimers bound at OR1 and OR2 to form octamers.
• These cooperative interactions increase the sensitivity of
regulation.
Figure 27.29: Ol3 and Or3 are brought into proximity by formation of the repressor octamer.
Transcription attenuation in the
leader of the E. coli trp operon
26.12 The trp Operon Is a Repressible
Operon with Three Transcription Units
• The trp operon is negatively
controlled by the level of its
product, the amino acid
tryptophan (autoregulation).
• The amino acid tryptophan
activates an inactive repressor
encoded by trpR.
• A repressor (or activator) will
act on all loci that have a copy
of its target operator sequence.
Figure 26.32: Operators may lie at
various positions relative to the
promoter.
26.13 The trp Operon Is Also Controlled by
Attenuation
• attenuation – The regulation of bacterial operons by
controlling termination of transcription at a site located
before the first structural gene.
Figure 26.33: Termination can be
controlled via changes in RNA secondary
structure that are determined by
ribosome movement.
26.13 The trp Operon Is Also Controlled by
Attenuation
• An attenuator (intrinsic
terminator) is located
between the promoter and
the first gene of the trp
cluster.
• The absence of Trp-tRNA
suppresses termination and
results in a 10 increase in
transcription.
Figure 26.34: An attenuator controls
the progression of RNA polymerase
into the trp genes.
26.14 Attenuation Can Be Controlled by
Translation
• The leader region of the trp operon has a 14-codon open
reading frame that includes two codons for tryptophan.
• The structure of RNA at the attenuator depends on
whether this reading frame is translated.
• In the presence of Trp-tRNA, the leader is translated to a
leader peptide, and the attenuator is able to form the
hairpin that causes termination.
26.14 Attenuation Can Be Controlled by
Translation
Figure 26.35: The trp operon has a short sequence coding for a leader peptide that is
located between the operator and the attenuator.
26.14 Attenuation Can Be Controlled by
Translation
Figure 26.36: The trp leader region can exist
in alternative base-paired conformations.
Figure 26.37: The alternatives
for RNA polymerase at the
attenuator depend on the
location of the ribosome.
26.14 Attenuation Can Be Controlled by
Translation
• In the absence of Trp-tRNA, the ribosome stalls at the
tryptophan codons and an alternative secondary
structure prevents formation of the hairpin, so that
transcription continues.
Figure 26.38: In the presence of
tryptophan tRNA, ribosomes translate
the leader peptide and are released.
Mechanism of transcription activation of the yeast GAL
genes.
A classical model of inducible gene
expression in a eukaryote.
• GAL1 galactokinase (mutant allele is gal1)
• GAL2 galactose permease
• GAL7 galactose uridyl transferase
• GAL10 galactose-glucose epimerase
• GAL4 positive regulator (mutant is gal4)
• GAL80 negative regulator (mutant is gal80)
Yeast GAL genes are positively regulated at
the level of transcription activation.
• A gal4, gal80 double mutant fails to induce
expression of galactose enzymes.
• Interpretation is that GAL4 targets the
structural genes to activate transcription.
• GAL80 interacts with GAL4 to control its
activity.
Deletion analysis of yeast GAL1 upstream region defined
an Upstream Activating Site (UASG).
GAL1-lacZ and GAL10-lacZ fusions used for deletion analysis.
UASG had many properties of the Enhancer defined in SV40.
Four copies of a nearly
symmetric 17 bp GAL4 site.
Experiments on the mechanism of
transcription activation by GAL4.
Separation of transcription activation
from DNA-binding function.
DNA-binding and transcription activation are
separable functions.
DNA-binding and transcription activation are
separable functions.
Current understanding of yeast
GAL gene activation
• GAL1-GAL80 complex DOES bind UASG DNA (a is wrong on this).
• GAL3 is the galactose sensor for GAL gene induction
• GAL3 is closely related to GAL1, also binds galactose and ATP
• GAL1 itself acts instead of GAL3 to bind GAL80 in Kluveromyces
lactis, the whey (skimmed milk) yeast, which hydrolyses lactose and
then converts the galactose to glucose
Deletion analysis of yeast GAL1 upstream region defined
an Upstream Activating Site (UASG) and Mig1 site.
Mig1 protein mediates glucose repression of GAL1
Mig1 recruits Ssn6 and Tup1
Mig1 also binds GAL4 promoter and lowers GAL4 protein levels
Four copies of a nearly
symmetric 17 bp GAL4 site.
Eukaryotic gene activators recruit
RNA polymerase to the
promoter.
• Activating regions associate with mediator which
in turn associates with RNA polymerase.
• Screen for yeast mutants with increased
activation by GAL4(1-100) identified a potentiator
mutant in GAL11, a component of Mediator.
• Gal11 when fused to LexA activates transcription.
This is an activator bypass experiment.
• Results favour the idea that GAL4 recruits
polymerase to the promoter by helping it bind as
CAP/CRP does in E. coli.
Uses of GAL4 Number 1: The yeast two hybrid system for
identification of interacting proteins.
Can be used on a genomic scale to test all proteins against all others. Target protein fused to
DNA-binding domain in cells of one mating type mated to a library of activating region fusion proteins.
Uses of GAL4 Number 2: GAL4 activates transcription from UASG in
Drosophila.
Widely used for targeted protein expression in Drosophila
(GAL4-UAS two-component system).
Induction of ectopic eyes by
GAL4-targeted expression of
the eyeless gene in Drosophila
The GAL4-UAS binary system is used to map brain
neurons involved in memories and behaviours and
to target gene expression there.
• rutabaga mutant flies lack an adenlyl-cyclase required for synaptic
plasticity and cannot learn a variety of training tasks.
• A UAS-RUTABAGA construct was expressed under the control of
different GAL4 drivers in a rutabaga mutant fly to see which brain
cells are needed to learn particular tasks.
• Mushroom body cells (somewhat similar to mammalian
hippocampus) need RUTABAGA protein at synapses to learn to
avoid particular odours and central complex cells need it for visual
learning.
• Drosophila connectome is all the neuron types and pathways in the
whole CNS
Liam.Keegan@ceitec.muni.cz
Lewin, GENES XI
Chapter 26
Operons, prokaryotic gene regulation and
yeast GAL4
Lewin’s GENES XII (PDF)
https://pdfroom.com/books/lewins-genes-xii/Wx5aDYKl2BJ
biotecher.ir
https://biotecher.ir/wp-content/uploads/2018/06/bio...
Showing that GAL4 binds the GAL UAS.
Methods for identifying and characterising
sequence-specific DNA-binding proteins.
DNase I protection assay to precisely define binding sites of
sequence-specific DNA binding proteins and to measure DNAbinding
affinity, (DNase I Footprinting)
The GAL4-binding site is CGG-N(11)-CCG.
The GAL4 DNA-binding domain has a Zn2Cys6
binuclear cluster structure found in over 60
proteins but restricted to fungi.
Since GAL4 has a type of DNA-binding domain not seen in animals we will discuss
DNA sequence recognition and more common types of DNA-binding domains tomorr
Molecular Genetics 3 Lecture 5 4.10.11
Summary
RNA polymerases and RNA pol II promoters.
TAFs
Transcriptional regulation in yeast.
Uses of GAL4
DNA looping is the simplest explanation for
GAL4 action.
Condensation and decondensation of chromatin in the cell cycle.
• Chromatin condenses during mitosis and
decondenses in interphase when most genes are
expressed.
• Histone deacetylases (HDACs) facilitate
chromatin condensation and contribute to
gene repression (Hda1, Rpd3).
• Histone acetyl transferases (HATs), like GCN5
in the SAGA complex, acetylate histone tails
and contribute to chromatin decondensation
and gene activation.
• Nucleosome remodelling complexes (Swi/Snf
complex), can remove nucleosomes from a
promoter region or deposit them there.
Imposing a requirement for nucleosome modifiers for
GAL1 activation.
Neither GCN5 histone acetyltransferase nor the nucleosome remodelling complex Swi/Snf
complex are required for GAL1 activation nor to activate 95% of yeast genes.
Nevertheless GAL4 recruits these activities to the promoter and they become necessary if
GAL4 sites are weakened or when chromosomes condense for mitosis.
Unusual chromosomes in Saccharomyces
cerevisiae
• The nuclear membrane was seen only after the electron microscope was
invented. The nuclear membrane does not break down at mitosis.
• The genome is small and divided among 16 chromosomes. S. cerevisiae
lacks a Histone protein 1 that binds the linker DNA between nucleosomes
and causes them to condense more. Cell division takes place without
formation of visible metaphase chromosomes.
• The fission yeast Schizosaccharomyces pombe has fewer chromosomes and
these become visible at metaphase. It is quite different from budding yeast
in DNA sequence and was promoted as being more like mammals by Paul
Nurse and others who used it to study the cell cycle, (“Schizophrenic
pombe”).
26.15 Stringent Control by Stable RNA
Transcription
• Poor growth conditions cause bacteria to produce the
small-molecule regulators (p)ppGpp.
• Stringent response – The ability of a bacterium to shut
down synthesis of ribosomes and tRNA in a poor growth
medium.
• The trigger for the reaction is the entry of uncharged tRNA
into the ribosomal A site.
• (p)ppGpp competes with ATP during formation of the open
complex during transcription initiation by RNA polymerase
and inhibits the reaction.
26.15 Stringent Control by Stable RNA
Transcription
• Relaxed mutants – In E.
coli, these do not display
the stringent response to
starvation for amino acids
(or other nutritional
deprivation).
• Stringent factor – The
protein RelA, which is
associated with ribosomes.
It synthesizes ppGpp and
pppGpp when an
uncharged tRNA enters the
ribosome.
Figure 26.40: Stringent factor catalyzes the
synthesis of pppGpp and ppGpp; ribosomal
proteins can dephosphorylate pppGpp to
ppGpp.
26.16 r-Protein Synthesis Is Controlled by
Autoregulation
• Translation of an r-protein operon can be controlled by a
product of the operon that binds to a site on the
polycistronic mRNA.
Figure 26.42: Translation of the r-protein
operons is autogenously controlled and
responds to the level of rRNA.
26.10 The Operator Competes with LowAffinity
Sites to Bind Repressor
• Proteins that have a high affinity for a specific DNA
sequence also have a low affinity for other DNA
sequences.
• Every base pair in the bacterial genome is the start of a
low-affinity binding site for repressor.
Figure 26.23: lac repressor binds strongly and specifically to its operator, but is released by
inducer. All equilibrium constants are in M–1.
26.10 The Operator Competes with LowAffinity
Sites to Bind Repressor
• The large number of lowaffinity
sites ensures that all
repressor protein is bound
to DNA.
• Repressor binds to the
operator by moving from a
low-affinity site rather than
by equilibrating from
solution.
Figure 26.24: Virtually all the repressor
in the cell is bound to DNA.
26.10 The Operator Competes with LowAffinity
Sites to Bind Repressor
• In the absence of inducer, the operator has an affinity for
repressor that is 107 times that of a low-affinity site.
• The level of 10 repressor tetramers per cell ensures that
the operator is bound by repressor 96% of the time.
• Induction reduces the affinity for the operator to 104
times that of low-affinity sites, so that operator is bound
only 3% of the time.
26.1 Introduction
• negative control of transcription
by a repressor removed by an
inducer (lac repressor + IPTG),
• positive control of transcription
by activator and inducer (CAP +
cAMP),
•
• (also possible, lower half,
• negative control of transcription by a
repressor and corepressor
• and positive control of transcription by
having an activator that is removed)
Figure 26.04: Regulatory circuits can be designed
from all possible combinations of positive and
negative control with inducible and repressible
control.
Spare slides!
DNA is superior to dsRNA as a genomic material because of the major groove.
Specific base recognition by proteins is easy in DNA.
dsRNA
A-helix
Deep and narrow
Major grrove.
DNA
B-form
Wide and
Accesible
Major groove.
Wide major groove means DNA sequence can be
recognized by DNA-binding proteins.
26.1 Introduction
• regulator gene – A gene that codes for a product
(typically protein) that controls the expression of other
genes (usually at the level of transcription).
• structural gene – A gene that codes for any RNA or
protein product other than a regulator.
Figure 26.01: A regulator gene
codes for a protein that acts at
a target site on DNA.
26.1 Introduction
• trans-acting – A product that can function on any copy
of its target DNA. This implies that it is a diffusible protein
or RNA.
• cis-acting – A site that affects the activity only of
sequences on its own molecule of DNA (or RNA); this
property usually implies that the site does not code for
protein.
26.1 Introduction
• Gene regulation in vivo
can utilize any of these
mechanisms, resulting in
all four combinations:
negative inducible,
negative repressible,
positive inducible, and
positive repressible.
Figure 26.04: Regulatory circuits can be
designed from all possible combinations
of positive and negative control with
inducible and repressible control.
Beginnings of genetics in
Saccharomyces cerevisiae.
• Yeast genetics began only in the 1950s - much later than E. coli genetics.
Commercial strains of brewer’s and baker’s yeast are stable diploids or
tetraploids and could not be studied genetically.
• Occasional meiosis in diploid stains gives an ascus containing four haploid spores
two with a and two with α mating types but these immediately mate back to
diploids (Ascomycete family of fungi). Pure haploid cultures could not be grown
from spores because after the first haploid cell division the mother cell switches
mating type and can mate with cell it just budded off.
• Yeast genetics required mutants in a gene called HO (homothallism – is a plant
term for having both types of sexual parts on one organism). The ho mutation
keep haploid cell lines stable because it prevents mating type switching. Winge,
(Carlsberg labs, Copenhagen), Lindegren, (Anheuser Busch, Carbondale, Illinois).
• Much of yeast genetics is done with the haploid strains that have only one copy
of each gene (same as E. coli) but can be mated to give diploids and sporulated
back to haploids by nitrogen starvation.
Genome sizes increase faster than gene numbers among
the main model organisms.
4,600 kb
13,500 kb 3x genome size of E. coli
165,000 kb 10X genome size of yeast
20X Drosophila genome
Bacterial minimum 1800.
Yeast has 6,000 genes. About 2X
basic eukaryotic minimum
set of 2,700 or so.
Simpler animals like Drosophila
have 2X as many genes as yeast.
Humans and other mammals
have only 2X Drosophila.
3,000,000 kb