Transcript Document

Molecular Mechanisms of Gene Regulation:
The Operon (Ch7)
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
The Lactose Operon
lacZ : b-galactosidase
lacY : lactose (galactoside) permease
lacA : galactoside transacetylase
Diauxic
growth
Bi-phasic;
cells grow
on one
carbon
source until
depleted &
then grow
on the
other
Francois Jacob
Jaques Monod
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).
Induction of
the lac operon
Negative Regulation of transcription
Inducible
Negative Regulation
Repressible
Positive Regulation
The lac Operon
The nature of the lac inducer
Complementation
1. 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
Complementation
using two
(recessive)
mutants
Interpretations
 very different
Mutant Repressor Gene
(no repressor made)
Lac
product?
+ inducer
- inducer
Y/N
Y/N
Y/N
Y/N
Conclusion: Both lac operons are repressible
recessive
Mutant Operator (Oc)
Lac
product?
Y/N
Y/N
Conclusion: One lac operon non-repressible
cis-dominant
Mutant Repressor Gene
(cannot bind inducer)
Lac
product?
Y/N
Y/N
Conclusion: Both lac operons are uninducible
cis and trans dominant
Mutant Repressor Gene
Lac
(cannot bind operator sequence) product?
Y/N
Y/N
Conclusion: Both lac operons are nonrepressible
dominant-negative
Repression & Activation
Binding between lac Operator & lac
Repressor
Non-metabolizable analogue of lactose
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)
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.
Effects of Mutations in the 3 lac Operators
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
-cAMP responds
to glucose conc.
ATP
Inhibited by
glucose
Adenylcyclase
- glucose uptake
lowers the
quantity of cAMP
by inhibiting the
enzyme
adenylcyclase.
Cyclic AMP
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
The Molecular Mechanism of
c-AMP-CRP Action
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
Rifampicin-inhibits RNA polymerase
Only if added before RNA polyermase has initiated
transcription  rifampicin resistant complex
+ rifampicin + nucleotides
+ rifampicin + nucleotides
Conclusion- cAMP-CRP (CAP) promotes open
promoter complex formation
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.
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.
DNA Looping
-cooperative binding between proteins to
remote sites
Measuring DNA bending
1. cut DNA fragment with different
restriction enzymes
2. Bind protein
Relationship between electrphoretic mobility
and bent DNA (w/protein)
Bend center  protein
binding site
DNA bending model for cAMP-CRP activation
-bend facilitates polymerase binding (exposes
promoter)
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
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)
-sulfated glycosoaminoglycan (chain)
-joints, vitreous humor
-viscosity increasing agent, anti-coagulant
-binds RNA polymerase inhibiting association
with promoter
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
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
Experiment:
1. Add RNA polymerase + lac promoter
(used fluorescent labeled UTP analog)
2. (1) no addition
(3) + repressor
(2) + heparin
(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
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!
Maltose Operon
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
malEp
The malEp & malKp region
(divergent operons)
-2 operons transcribed in opposite directions
(3 genes each)
-3 CRP binding sites & 5 MalT binding sites
The MalT Binding Sites
-each site consists of 2 6-bp overlapping
binding regions
-the third site
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.
Arabinose Operon
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
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
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.
Map of the ara Control Region
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
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
Experimental Evidence of Looping
1. Observed by electron microscopy
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
Binding of AraC to O2 site
-in mutant O2 site, dissociation of AraC from
site occurred at faster rate than WT.
Binding of AraC to I site
Addition of Arabinose Breaks Loop
between araO2 and araI
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
AraC autoregulates its Own
Transcription
araC
……
araO2
araI
I1 I2
araO1
araPBAD
araPc
Note: presumably
this can occur +/arabinose (with
control region
looped or unlooped).
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.
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.
Tryptophan
Operon
Tryptophan
biosynthesis
(anabolic
pathway)
- 5 structural
genes (a-e)
- promoter/
operator
region (p,o)
-regulator gene
(trpR)
Tryptophan: Effect on Negative Control
Low Tryptophan  no repression
Repression: tryptophan is a co-repressor 
binds (inactive) apo-repressor converting it
to active repressor
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 protein’s 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
Attenuator Region of Trp Operon
…
…
Low tryptophan: transcription of trp operon
genes RNA polymerase reads through
attenuator.
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.
3. The inverted repeat forms a hairpin loop.
4. When RNA polymerase reaches string of
U’s…
…the polymerase pauses, the hairpin forms
 Transcript is released
 Termination occurs before transcription
reaches the trp (structural) genes
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
Mechanism of Attenuation
…
…
Key insight: mRNA produced from attenuator region
can fold into two different secondary structures
Stem loops: 1-2, 3-4
Stem loop: 2-3
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?
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
1. Recall that in bacteria, translation typically
occurs almost simultaneously with
transcription.
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
The stalled ribosome prevents the formation
of stem loops 1-2/3-4 and promote the
formation of stem loop structure 2-3
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
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.
Effect on
ribosome and
transcription
at HIGH Trp
levels
Note: the 14
amino acid
leader peptide
is synthesized
-This mechanism involves: transcriptionaltranslational coupling.
-Relies on rate of transcription & translation
to be comparable  if RNA polymerase >>
ribosome, it might pass through attenuator
region before ribosome had a chance to stall
at the tryptophan codons.
The Trp Operon of Bacillus subtilis
-mRNA secondary structure controlled by TRAP
not by ribosome
1. Attenuation response controlled by trp RNAbinding attenuation protein (TRAP)
2. Protein assists in translational termination.
Absence of trp
transcription
proceeds
2. Trp-TRAP binds leader sequences by recognizing 11
triplet codons.
3. Blocks anti-termination formation.
4. Allows
formation of
termination
loop
5. Result:
translational
termination
occurs
1. TRAP binds
11 tryptophan
residues.