Download POSITIVE REGULATION – THE ARA OPERON

POSITIVE REGULATION – THE ARA OPERON
-first example of positive regulation; Englesberg and colleagues
-met with much opposition as it was thought that the wellcharacterized negative regulation of the lac system was
representative of ALL gene regulation
-ara operon encodes genes responsible for utilization of Larabinose
-L-arabinose is converted to D-xylulose-5-phosphate, which can
then be used by other pathways
-3 structural genes; araB, araA, araD
-araBAD are induced in presence of arabinose, much like lac genes
are induced in presence of lactose
-one regulatory gene transcribed in opposite direction; araC
-AraC functions as an activator upon binding inducer arabinose by
binding at araI and stimulating transcription
-AraC also functions as a repressor in absence of inducer arabinose
by binding to two operators (araO2 and araO1) and preventing
transcription through DNA looping
Identification of AraC
-identified 15 mutants that are all non-inducible Ara-all failed to complement each other so must be in same gene –
araC
-all are recessive to araC+ (NB. Very different from lac!)
-these mutants contained nonsense, missense, and deletion
mutations in araC
Lac operon
-expression induced by lactose
-lacI-= constitutive Lac+
-lacI-/lacI+ = Lac+; recessive
Ara operon
-expression induced by
arabinose
-araC- = non-inducible Ara-araC-/araC+ = Ara+; recessive
-suggests araC- mutations knock out a POSITIVE function
required for expression of araBAD
Isolation of araCc alleles
-constitutive mutants are very rare, suggesting that they do not
inactivate the regulator AraC (reverse for LacI)
-isolated using D-fucose
-D-fucose binds AraC and prevents it from binding arabinose,
thereby preventing induction of araBAD operon
-wildtype E. coli can not grow on minimal arabinose plates
containing fucose
-mutants that constitutively express ara operon WILL grow
-mutants all mapped to araC
-araCc are dominant to araC-
Identification of araI
-critics believed that araC- mutations acted by interfering with a
negative regulatory system
-eg. they suggested that araC- mutations prevented accumulation
of inducer in the presence of arabinose, which would alleviate
repression
-to try and identify a negative regulator, Englesberg et al. looked
for suppressors of an araC deletion allele, araC∆719
-mutants were rare and all mapped to a site, araI, near, but not in,
the araBAD genes
-these mutations identify an AraC binding site necessary for
inducible activation of araBAD expression
Definitive Proof of Positive Regulation by AraC
-not until several years later
-an in vitro coupled transcription-translation system was developed
-DNA encoding araB and control region + RNAP + ribosomes
-require addition of AraC to get AraB synthesis
-fucose blocks AraB synthesis
-AraCc obtained from a fucose resistant strain is insensitive to
fucose inhibition
-requires RNAP binding site and araI
-araIc mutant DNA does not require AraC for AraB synthesis
AraC is also a Repressor!
-if AraC is solely an activator, then AraCc should be dominant to
wildtype AraC
Genotype
araC
araCaraCc
F’araC+/araCF’araCc/araC-
Phenotype
Inducible Ara+
Non-inducible AraConstitutive Ara+
Inducible Ara+
Constitutive Ara+
F’araC+/araCc
Inducible, Ara+
+
-what’s going on?
-hypothesize that AraC can exist in 2 states, P1 and P2
-P1 – in absence of arabinose; functions as a repressor
-P2 – in presence of arabinose; functions as an activator
-AraCc mutant is locked in P2 form, but in a merodiploid F’
araC+/araCc, wildtype AraC represses expression in absence of
arabinose, preventing activation by AraCc
Identification of an Operator for the ara operon
-deletions that extended beyond araC caused increased basal
expression of the araBAD operon
-propose that deletion removes an operator
-now know that AraC binds to three sites in the araBAD regulatory
region, araI, araO1, and araO2
-in absence of arabinose, AraC binds araO2 and araI and represses
araBAD transcription
-arabinose alters AraC such that repression is alleviated and
transcription is initiated (requires a second activator, CAP)
-in addition, AraC binds to araO1 in absence of arabinose to
repress its own transcription
CAP/CRP
-Catabolite Activator Protein (CAP) or Catabolite Repression
Protein (CRP)
-transcription activator that controls expression of genes involved
in carbon and energy source utilization
-since glucose is the preferred carbon source of E. coli, CAP
ensures that other carbon utilization pathways are not expressed in
the presence of glucose à CATABOLITE REPRESSION
-accordingly, CAP functions as a transcription activator of genes
involved in metabolism of alternative carbon sources when glucose
is NOT present (ie. lac, ara, gal, mal operons)
-it does this by sensing the levels of cAMP (cyclic AMP)
-when glucose is being metabolized, levels of cAMP (cyclic AMP)
are decreased
-conversely, when glucose is not being metabolized levels of
cAMP are elevated
-CAP is a symmetrical dimer of two identical subunits
-when bound to cAMP (low glucose, high cAMP), CAP is active
and binds to a specific palindrome found upstream of genes that
are controlled by catabolite repression
-consensus: 5’-AAATGTGATCT-AGATCACATTT-3’
-DNA binding mediated by a HTH present in each subunit and
leads to DNA bending (crystal structure)
Different CAP promoters
-CAP-dependent promoters can be grouped into 3 classes based on
location of the CAP binding site
i)
Class I
-require only CAP for transcription activation
-CAP binding site is upstream of RNAP binding site
-binding site can be located at various distances from start site as
long as it is on the same face of the helix as the RNAP binding site
(-61.5, -72.5, -82.5, -92.5)
-protoype is lacP1 promoter
ii) Class II
-require only CAP for transcription activation
-CAP binding site overlaps RNAP binding site
-replaces –35 binding determinants for RNAP
-prototype is galP1 promoter (CAP binding site at –41.5)
iii) Class III
-require a regulon-specific activator in addition to CAP for
transcription activation
-CAP binding site usually located more than 90 bp from
transcription start site
-eg. araBAD promoter
-much work suggests that the mechanisms of transcription
activation vary at Class I, Class II, and Class III promoters
Transcription Activation at Type I Promoters
-determinants within CAP and RNAP that are essential for
transcription activation specifically at Type I promoters have been
identified
1) CAP determinants for transcription activation
-mutants were sought which were specifically defective in
transcription from Class I promoters, but not in DNA binding or
DNA bending à POSITIVE CONTROL MUTANTS
-random mutagenesis of gene encoding CAP, in vivo screen for
loss of ability to activate transcription at Class I promoter, but
retention of ability to bind DNA (still represses)
-mutants are designated crppc (positive control)
-all localize to a region that is surface exposed in the crystal
structure à aa 156-162 (ACTIVATING REGION I, ARI)
-purified mutant proteins all bind and bend DNA but fail to
activate transcription of Class I promoters
-therefore, an additional step after DNA binding necessary to
activate transcription that requires AR1
2) RNAP determinants for transcription activation
-deletion of or point mutations in the C-terminal domain of the α
subunit preclude CAP-mediated activation at Class I promoters,
although the RNAP molecules are still able to bind DNA and
transcribe from an un-regulated promoter
-anti-α monoclonal antibodies inhibit CAP-mediated activation at
Class I promoters, but only partially inhibit CAP-independent
transcription
-aa 261, 265 and 270 essential for CAP-mediated transcription
-mutations alter a tightly folded C-terminal domain that is joined to
an N-terminal domain by a flexible linker
-hypothesize this region plays a role in AR1 activation
i)
Mechanism of transcription activation
-transcription activation at Class I CAP-dependent promoters
requires a protein-protein contact between AR1 and the C-terminus
of the a subunit of RNAP
-since CAP functions as a symmetrical dimer, which AR1 region is
required? Both?
-test using ORIENTED HETERODIMER EXPERIMENT
-CAP heterodimers are constructed that carry one subunit that has
altered DNA binding specificity à V181 (but functional in
transcription activation); and one subunit defective in AR1 à A158
(but having wild-type DNA binding specificity)
-the subunits are oriented on the promoter by using CAP binding
sites that are specific for the altered DNA binding mutant in one
half of the palindrome à L29 or L8
-by determining transcription levels mediated by different
combinations of dimers and binding sites can determine which
AR1 region is important
-this approach determined that only the promoter proximal AR1
region is important for transcription activation at Class I CAPdependent promoters
Transcription Activation at Type II Promoters
-transcription activation requires 2 distinct protein-protein
interactions between different areas of CAP and RNAP that affect
different stages of transcription initiation
i)
A second activating region in CAP
-a screen was performed to determine if CAP contains a second
AR required for activation at Class II CAP promoters
-random mutagenesis of CAP à screen for mutants specifically
defective in activation of Class II genes
-isolated mutations in His-19, His-21, and Lys-101
-all are next to each other in crystal structure, all are positively
charged à AR2
-positive charge of AR2 is required for transcription activation of
Class II promoters
-these mutations affect transcription activation at Class II, but not
Class I CAP-dependent promoters
-they have no effect on DNA binding or bending
ii) Identification of residues in RNAP that interact with AR2
-mutations at amino acids 162-165 of rpoA (N-terminal domain of
α subunit) yield RNAP that is defective in transcription activation
at Class II promoters, but not Class I, or unregulated promoters
-these amino acids are all negatively charged, suggesting they
might interact with the positively charged AR2
-in complex of CAP, RNAP, and DNA, AR2 and aa 162-165 of α
are located next to one another
iii) Mechanism of transcription activation at Class II promoters
-transcription activation at Class II promoters involves an
interaction between AR2 and the NTD of the α subunit of RNAP
-which AR2 in the dimer is required à ORIENTED
HETERODIMERS
-AR2 in the downstream CAP subunit of the dimer is required for
transcription activation at Class II promoters
-NB à AR1 is also required at Class II promoters
-because Class II promoters don’t contain an optimal –35,
formation of the closed complex is inhibited
-binding between AR1 and the CTD of a overcomes this inhibition
Class II promoters require 2 separate interactions between
CAP and RNAP
-AR1 enhances formation of closed complex, AR2 stimulates open
complex formation
Other Characterized Activator-RNAP Contacts
-protein-protein contact between activators and RNAP is a
common requirement for transcription activation
α
-transcription factors which bind upstream of the promoter require
α CTD to activate transcription
-RNAP carrying an α subunit with a deletion of the CTD no longer
facilitates transcription from promoters of genes requiring some
activator proteins
-eg. PhoB, OmpR, OxyR
β’
-ssDNA binding protein of bacteriophage N4 stimulates
transcription of late phage genes by interacting with a conserved
domain of β’
σ
-lcI encodes dual function activator/repressor responsible for the
lytic/lysogenic growth decision in λ
-mutants in an N-terminal acidic patch are specifically defective in
transcription activation, however still bind DNA normally
-suppressor mutations in σ restore activation of transcription to
these mutants
DEBATE OVER THE MECHANISM
-2 possible mechanisms of transcription activation through proteinprotein contact with RNAP:
1) recruitment of RNAP/stimulation of closed complex formation
2) stimulation of conformational changes in RNAP that facilitate
transcription initiation (AR2 of CAP)
-several experiments suggest that ANY protein-protein contact
between a DNA binding protein located upstream of a promoter
and RNAP will facilitate transcription, arguing that (I) is correct
-eg. if the C-terminal dimerization domain of lcI is fused to the
NTD of α in place of the native CTD, lcI can function as an
activator of transcription if a binding site is placed upstream of a
promoter
COMPLEX ACTIVATION
-transcription activation involving more than one activator protein
-often, one will be a GLOBAL REGULATOR that controls
expression of large numbers of genes in response to a global
metabolic signal, while the other will be a SPECIFIC
REGULATOR, triggering expression of a small number of genes
in response to a specific inducer molecule
-several mechanisms of complex regulation:
I.
nucleoprotein structures
-binding of multiple activators forms a nucleoprotein complex that
triggers transcription activation
II. repositioning
-binding of a second activator repositions an activator that is
already bound to DNA, such that it is now in position to activate
transcription
III. simultaneous touching
-multiple activators interact with distinct regions of RNAP to
facilitate transcription activation
-common with regulators that interact with σ70
IV. recruitment of bystanders/DNA looping
-IHF or other DNA bending protein facilitates contact of RNAP
with transcription factors bound far upstream of the promoter
-eg. σ54 promoters