Molecular Mechanisms of Gene Regulation:

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Molecular Mechanisms of Gene Regulation The
Operon (Ch7)
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Operon- set of genes that are coordinately
controlled by a regulatory protein AND
transcribed as a single polycistronic message
Regulon- set of related genes that are
transcribed as separate units but are controlled
by the same regulatory protein
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The Lactose Operon
lacZ b-galactosidase lacY lactose
(galactoside) permease lacA galactoside
transacetylase
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Diauxic growth
Bi-phasic cells grow on one carbon source until
depleted then grow on the other
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Francois Jacob
Jaques Monod
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1. Diauxic growth is dependent upon the carbon
(sugar) source used.
2. In E. coli two classes of sugar sources (i)
glucose, mannose, fructose (ii) lactose, maltose
3. Growth on class (i) combinations, i.e. glucose
mannose ? no diauxic growth same with class
(ii) mixtures.
4. Diauxy is observed when cells are grown in
mixtures containing (i) (ii).
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Induction of the lac operon
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Negative Regulation of transcription
Inducible
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Negative Regulation
Repressible
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Positive Regulation
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The lac Operon
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The nature of the lac inducer
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Complementation

• Restoration of phenotype

2. Different types genetic material
3. Mutation with phenotype ? add DNA (gene
product) ? restores phenotype
Typical conclusion mutation complementing DNA
encode-for or are the same gene
Alternate conclusions compensatory affects
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Complementation using two (recessive) mutants
Interpretations ? very different
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Mutant Repressor Gene
Lac product? inducer - inducer
(no repressor made)
Y/N
Y/N
Y/N
Y/N
Conclusion Both lac operons are repressible
recessive
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Mutant Operator (Oc)
Lac product?
Y/N
Y/N
Conclusion One lac operon non-repressible
cis-dominant
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Mutant Repressor Gene (cannot bind inducer)
Lac product?
Y/N
Y/N
Conclusion Both lac operons are uninducible
cis and trans dominant
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Mutant Repressor Gene
Lac product?
(cannot bind operator sequence)
Y/N
Y/N
Conclusion Both lac operons are non-repressible
dominant-negative
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Repression Activation
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Binding between lac Operator lac Repressor
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Non-metabolizable analogue of lactose
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The lac control region
1. 3 operators (O1, O2, O3) region where
regulatory proteins bind
2. RNA polymerase binding site (promoter)
3. cAMP-CRP complex binding site (CAP)
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b-Galactosidase Activity
1. Recall that the first gene in the lac operon
is lacZ (b-galactosidase)
2. Enzyme activity can easily be measured using
X-Gal or p-nitrophenol-galactoside (colorimetric
assays that can be quantified)
3. Therefore effects on regulation can be
monitored by measuring b-galactosidase activity.
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Effects of Mutations in the 3 lac Operators
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Positive Control of the lac Operon
1. Removal of repressor is NOT enough to activate
the operon.
2. The lac operon has a mechanism for reponding
to glucose levels.
Why? (i) When glucose levels are high, the cell
wants to repress transcription of other operons
(lactose)
(ii) When glucose levels are low
lactose present ? upregulate lac operon
? Catabolite repression selection in favor of
glucose metabolism
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-cAMP responds to glucose conc.
ATP
Inhibited by glucose
Adenylcyclase
- glucose uptake lowers the quantity of cAMP by
inhibiting the enzyme adenylcyclase.
Cyclic AMP
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1. Addition of cAMP overcomes catabolite
repression.
2. The activator is a complex between cAMP and a
protein catabolite activator protein (CAP) aka
cAMP receptor protein (CRP) ? gene crp.
3. A mutant CRP protein with 10 lower affinity
for cAMP if cAMP-CRP complex important for
activation then mutant should have reduced
production of b-galactosidase
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The Molecular Mechanism of c-AMP-CRP Action
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1. cAMP-CRP complex stimulates transcription by
binding to (activator) site adjacent to promoter.
2. cAMP-CRP recruits and helps RNA polymerase to
bind to the promoter.
3. Recruitment has two steps
-formation of closed promoter complex
-conversion of closed promoter complex to open
promoter complex
?increases rate of open promoter complex formation
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Rifampicin-inhibits RNA polymerase
Only if added before RNA polyermase has initiated
transcription ? rifampicin resistant complex
rifampicin nucleotides
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rifampicin nucleotides
Conclusion- cAMP-CRP (CAP) promotes open promoter
complex formation
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How does cAMP-CRP binding to the activator site
facilitate binding of polymerase to the promoter?
1. cAMP-CRP complex touches the polymerase ?
cooperative binding
2. cAMP-CRP causes the DNA to bend.
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Direct Interaction Model
Evidence
(1) co-sedimentation (2) chemical cross-linking
(3) Dnase footprinting (4) mutations in CRP that
decrease activation but NOT DNA binding ?
interface that interacts with polymerase.
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DNA Looping
-cooperative binding between proteins to remote
sites
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Measuring DNA bending
1. cut DNA fragment with different restriction
enzymes
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2. Bind protein
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Relationship between electrphoretic mobility and
bent DNA (w/protein)
Bend center ? protein binding site
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DNA bending model for cAMP-CRP activation
-bend facilitates polymerase binding (exposes
promoter)
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Mechanism of Repression
1. Assumption repressor blocks polymerase access
to promoter.
2. Experimental evidence, however, has shown that
RNA polymerase can STILL bind to promoter in the
presence of repressor
Rifampicin ?no transcription unless open promoter
complex has formed
Experiment 1 DNA, polymerase, repressor
? add inducer, nucleotides, rifampicin
Result
Transcription occurred ? repressor had not
prevented formation of open complex
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Experiment 2
1. DNA repressor (5-10 min)
2. RNA polymerase (20 min)
3. Add heparin
-Blocks any further complex formation
all reaction components except CTP
4. Add CTP /- inducer (IPTG)
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-sulfated glycosoaminoglycan (chain)
-joints, vitreous humor
-viscosity increasing agent, anti-coagulant
-binds RNA polymerase inhibiting association with
promoter
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Further evidence showed that repressor and
polymerase can bind together to lac operator.
If lac repressor does not inhibit transcription
of the lac operon by blocking access to promoter,
how does it function?
Alternate theory repressor locks RNA polymerase
into a non-productive state.
Evidence formation of abortive transcripts
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HOWEVER
More recent studies have shown that
repressor/polymerase operator interactions are
in equilibrium.
Ratio of polymerase-promoter complex and free
polymerase/free promoter
And that previous experiments were simply
shifting or locking this equilibrium association
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Experiment
1. Add RNA polymerase lac promoter
(used fluorescent labeled UTP analog)

• (1) no addition (2) heparin
• (3) repressor (4) no DNA

Analysis (i) heparin known to prevent polymerase
(re)-association
(ii) If repressor does not block access to
polymerase it should not inhibit polymerase
association with promoter
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Result both heparin and repressor inhibits
(re)-association of polymerase with promoter.
Analysis (1) heparin binds polymerase preventing
association with DNA (2) repressor
does the same by binding to the operator adjacent
to the promoter and blocking access to the
promoter by RNA polymerase.
Conclusion Original competition hypothesis may
be correct!
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Maltose Operon
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1. mal regulon regulated by CRP
2. MalT also regulates the mal promoters
-requires ATP -activated by inducer
(maltotriose) -Some mal promoters malEp malKp
use both CRP and MalT
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The malEp malKp region
(divergent operons)
malEp
-2 operons transcribed in opposite directions (3
genes each)
-3 CRP binding sites 5 MalT binding sites
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The MalT Binding Sites
-each site consists of 2 6-bp overlapping binding
regions
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-the third site
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DNA footprinting showing 3-bp shift in MalT
binding after CRP (CAP) binding
-MalT has higher affinity for sites 3, 4, and 5
than for sites 3, 4, and 5.
-sites 3,4, and 5 are exactly 3-bps short of
maximal spacing for promoting RNA polymerase
binding.
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Arabinose Operon
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DNA Looping
-protein with DNA binding domain (yellow)
protein-protein interaction domain (blue)
-loop occurs if proteins can interact because
intervening sequence can loop out without
twisting
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1. insertions which disrupt the ability of the
proteins to bind to the same face of DNA inhibit
loop formation
-one double helical turn ? 10.5 bp
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1. Arabinose operon consists of 4 genes, 3
together transcribed in one direction (araPBAD),
the fourth araC ?divergent (araPc)
2. AraC is the control protein, acts as repressor
or activator depending upon binding conditions.
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Map of the ara Control Region
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Absence of Arabinose
Negative control- monomers of AraC bind to O2 and
I1 looping out the intervening sequence (210 bp)
blocking access to the promoter by RNA
polymerase
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Positive Control
1. Arabinose binds to AraC ?results in
conformational change in AraC.
2. Arabinose-AraC complex preferentially binds to
I2/I1 sequences (over O2/I1 sequence)
3. Promoter accessible to RNA polymerase
4. cAMP-CRP present (glucose absent) ? bind to Pc
site ?transcription stimulated
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Experimental Evidence of Looping
1. Observed by electron microscopy
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2. Looped DNA migrates differently than unlooped
on agarose gel.
-competition experiment (labeled) DNA AraC
-add excess unlabeled DNA
-can use info to determine ½ life of protein-DNA
interaction
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Binding of AraC to O2 site
-in mutant O2 site, dissociation of AraC from
site occurred at faster rate than WT.
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Binding of AraC to I site
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Addition of Arabinose Breaks Loop between araO2
and araI
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Notes on Regulation of the Arabinose Operon
1. Looping/unlooping is reversible. Add AraC ?
loop forms, add arabinose ? loop breaks, remove
arabinose (dilution) ? loop reforms (in presence
of AraC
2. AraC contacts I2 in the unlooped state but not
in the looped complex.
3. A single dimer of AraC is sufficient for loop
formation
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AraC autoregulates its Own Transcription
araC
araO2
araPc
araO1
Note presumably this can occur /- arabinose
(with control region looped or unlooped).
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Conclusions
I. Maltose Operon. 1. Mal operon controlled by
CRP MalT (transcription factor)
2. CRP stimulates transcrption by shifting MalT
from one set of binding sites to another (only 3
bp away)
3. Initial binding site of MalT is poorly aligned
with (enhancing transcription from) the promoters
4. The secondary sites are better aligned with
respect to the promoters and hence can facilitate
transcription.
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I. Arabinose Operon. 1. Ara operon controlled by
AraC.
2. AraC rpresses operon by looping out the DNA
between sites araO2 and araI1 (210 bp apart)
3. Arabinose derepresses the operon by causing
AraC to loosen its attachment to araO2 and to
bind to araI2 instead.
(beaks loop, allowing transcription)
4. cAMP-CRP further stimulates transcription by
binding to a site upstream of araI.
5. AraC regulates its own transcription by
binding to araO1 and preventing (leftward)
transcription of the araC gene.
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Tryptophan Operon
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Tryptophan biosynthesis
(anabolic pathway)
- 5 structural genes (a-e)
- promoter/ operator region (p,o)
-regulator gene (trpR)
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Tryptophan Effect on Negative Control
Low Tryptophan ? no repression
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Repression tryptophan is a co-repressor ? binds
(inactive) apo-repressor converting it to active
repressor
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1. Operator site lies within the promoter
2. Allosteric transition
Allosteric protein-protein whose shape is changed
upon binding of a particular molecule ? In the
new conformation the proteins ability to react
to a second molecule is altered
3. Trp operon has another level of control ?
attenuation
4. Repressor lowers transcription 70-fold (as
compared to derepressed state) ? attentuation
permits another 10-fold control ? total dynamic
range of control 700-fold
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Attenuator Region of Trp Operon


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Low tryptophan transcription of trp operon
genes? RNA polymerase reads through attenuator.
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High tryptophan attenuation, premature
termination ? attenuator causes premature
termination of transcription
1. Attenuator region contains transcription stop
signal (terminator) ? not STOP codon!
2. The terminator consists of an inverted repeat
followed by string of eight A-T pairs.
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3. The inverted repeat forms a hairpin loop.
4. When RNA polymerase reaches string of Us
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the polymerase pauses, the hairpin forms
? Transcript is released
? Termination occurs before transcription reaches
the trp (structural) genes
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Attenuation gives some insight into how the
operon is shut down, but how does the cell
activate trp operon expression (i.e. defeat
attenuation)?
?preventing hairpin formation would destroy
termination signal ? transcription would proceed
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Mechanism of Attenuation


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Key insight mRNA produced from attenuator region
can fold into two different secondary structures
Stem loops 1-2, 3-4
Stem loop 2-3
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1. Formation of stem loop structures 1-2 and 3-4
is more stable and results in the formation of a
termination (hairpin loop) structure/signal.
2. Formation of stem loop structure 2-3 would
result in the disruption of stem loops 1-2/3-4.
3. The stem loop structure formed between 2-3
does not result in termination signal ?
transcription would proceed.
Q. becomes How does the less stable structure
(stem-loop 2-3) form?
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The Importance of the Leader Region
-the 14 amino acid peptide formed from the leader
sequence has 2 tryptophans.
-trp is a rare amino acid
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1. Recall that in bacteria, translation typically
occurs almost simultaneously with transcription.
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2. Thus, as soon as trp leader region is
transcribed, translation begins.
Consider LOW Trp Conditions
3. During low tryptophan concentration, ribosome
will stall at trp sites.
4. The trp site is right in the middle of region
1 of the attenuator
? Meanwhile RNA polymerase continues to transcribe
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The stalled ribosome prevents the formation of
stem loops 1-2/3-4 and promote the formation of
stem loop structure 2-3
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1. Stem loop structure 2-3 does not result in
transcriptional termination ? whole operon mRNA
made.
2. What happens to the stalled ribosome?
(i) Since the genes in the operon have their own
start sites other ribosomes can come and
translate those proteins
(ii) Stalled ribosome can eventually either
incorporate trp-tRNA ( 3 more a.a. before
reaching stop codon) or dissociate from mRNA
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At HIGH Trp Conditions
1. When high levels of Trp-tRNA are present the
two tryptophan codons do not represent a barrier
translation ? ribosome breezes through.
2. Ribosome continues through element 1 (no
stalling) and reaches stop signal (UGA)
3. With no ribosome ? stem loops 1-2/2-3 form on
the mRNA ? halting transcription before
polymerase has chance to reach trp structural
genes.
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Effect on ribosome and transcription at HIGH Trp
levels
Note the 14 amino acid leader peptide is
synthesized
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-This mechanism involves transcriptional-translat
ional coupling.
-Relies on rate of transcription translation to
be comparable ? if RNA polymerase gtgt ribosome, it
might pass through attenuator region before
ribosome had a chance to stall at the tryptophan
codons.
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The Trp Operon of Bacillus subtilis
-mRNA secondary structure controlled by TRAP not
by ribosome
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1. Attenuation response controlled by trp
RNA-binding attenuation protein (TRAP)
2. Protein assists in translational termination.
Absence of trp transcription proceeds
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2. Trp-TRAP binds leader sequences by recognizing
11 triplet codons.
3. Blocks anti-termination formation.
1. TRAP binds 11 tryptophan residues.
4. Allows formation of termination loop
5. Result translational termination occurs