The arabinose operon regulation

March 2019
 Arabinose
• E. coli can use arabinose as a carbon source.
• It does this by converting arabinose into
xylulose-5-phosphate.
• The latter is an intermediate in the pentose
phosphate pathway.
• This requires the enzymes arabinose
isomerase, ribulose kinase, and ribulose-5-
phosphate epimerase.
• Arabinose isomerase is encoded for by the
araA gene.
• Ribulose kinase is encoded for by araB gene.
• Ribulose-5-phosphate epimerase is encoded
by araD gene.
• Transport of L-arabinose into cells requires 2
operons.
• These are araE and araFGH.
• Each capital letter in front of the letters ara,
represents a gene.
• araE gene encodes a protein that is involved in
low-affinity transport of L-arabinose.
• araFGH loci encode proteins involved in high
affinity transport of L-arabinose.
• araBAD code for three structural genes.
• araC operons specifies the regulatory gene.
• The araBAD genes consists of 3 structural genes.
• These are involved in the metabolism of
arabinose.
• araA codes for L-arabinose isomerase.
• It catalyzes the conversion of L-arabinose to L-
ribulose.
• araB codes for a ribulosekinase.
• This catalyzes the phsophorylation of L-
ribulose to L-ribulose-5-phosphate.
• ATP is the phosphate donor for the reaction.
• araD codes for L-ribulose-5-phosphate
epimerase.
• This catalyzes the conversion of LR5P to D-
xylulose-5-phosphate.
• The order of expression is BAD.
• The regulatory operon for araBAD has a single
structural gene.
• This is araC located to the left of the araBAD
operon.
• The araC gene is transcribed from its own
promoter in the opposite direction from the
araB, A, and D genes.
• The araC protein binds to a number of control sites as a
dimer.
• These are:
• araI2, araI1, and araO1 and araO2.
• The araC protein often exists as a tetramer because the
dimers interact with each other as they bind to the
multiple sites.
• The araC protein has an important characteristic.
• It can function both as an activator or a repressor of
transcription.
• It acts as a repressor in the absence of L-
arabinose.
• In the presence of L-arabinose, it acts as an
activator because the sugar binds to it and
changes its conformation.
• The activator form of araC can also block
transcription of the araC operon when high
concentration of the activator are present.
• This form of regulation is called
autoregulation.
• Most of the control sites for both araC and
araBAD genes are located between the araC
and araBAD loci.
• For araBAD genes the following are the
control sites:
• (i) araP1 which is the promoter.
The araBAD operon and its control sites:
Structural genes are coded for by araA, araB
and araD.
araC codes the regulatory protein and within
Its gene sequence the O2 site is found.
O2 is expressed by the P2 promoter.
• (ii) initiator site araI2.
• (iii) initiator site araI1.
• (iv) a site for the binding of CRP bound to cAMP
called araCRP.
• The araCRP is also known as the CAP binding site.
• (v) an operator site araO1.
• (vi) a second operator site located in the araC
gene about 100 bp from the start site.
• Transcription is modulated by CAP-cAMP
complex as in the lac system.
What happens when L-arabinose is absent?

• The araC repressor binds to araO2 and araO1

and the dimers associate with each other.
• This prevents/inhibits the expression of araC
gene because the RNAP is unable to pass the
araO2 site.
• In other words only a segment of the araC
protein is made in this situation.
• Meanwhile the expression of araBAD requires
the binding of a CRP-cAMP complex at the
araCRP binding site.
• Expression also requires the conversion of the
repressor into an activator and its binding at
araI1 and araI2.
• When the activator (araC) is absent there is no
transcription from araP1 promoter.
• Also in similar manner when araC protein is
absent, there is no transcription from araP1
even when the sugar L-arabinose is present.
• This really means one thing:
• That araC acts as a repressor and activator
and the 2 are alternative forms of the same
protein.
• The araBAD operon is negatively regulated by
the araC repressor and yet positively regulated
by the araC activator version of the same
protein if L-arabinose is present in the
medium to induce the necessary
conformational change.
• When L-arabinose is absent, the araC protein (as a
repressor) binds to both araO2 and araI1.
• This blocks transcription of araC at araO2.
• Approximately 100 nucleotides of araC are
transcribed before blockage occurs.
• When L arabinose is added, the araC becomes an
activator.
• It disengages from araO2 and associates with
araI2.
• The complete transcription of araC from araP2
is allowed to happen.
• This also allows the transcription from araP1
to occur.
• The role of AraC protein in the regulation of this
system is complex.
• First, it regulates its own synthesis.
• It does this by binding at araO1 and repressing
transcription of the araC gene when its
concentration exceeds about 40 copies per cell.
• Second, it acts as both a positive and a negative
regulator of the araBAD genes.
• It does this when it binds to ara02 and araI.
• This regulation can be summarized in the form
of four metabolic scenarios:
• (1) Glucose is abundant and arabinose is not.
• Under these conditions, the AraC protein
bound to ara02 and that bound to araI bind to
each other, forming a DNA loop of about 210
base pairs.
• In this configuration the system represses
transcription from the promoter for the
araBAD genes.
• (2) Glucose is not present (or is at low levels)
but arabinose is available.
• Under these conditions, CAP-cAMP becomes
abundant and binds to its site adjacent to araI.
• Arabinose also binds to the AraC protein,
altering its conformation.
• The DNA loop is opened, and the AraC protein
bound at araI now becomes an activator.
• Arabinose acts together with CAP-cAMP to
induce transcription of the araBAD genes.
• (3) Arabinose and glucose are both abundant.
(4) Arabinose and glucose are both absent.
• For both (3) and (4), the status of the system is
not entirely clear, but it remains repressed in
both cases.
• The ara operon is a complex regulatory system
that provides rapid and reversible responses
to changes in environmental conditions.
 Mutations in the ara operon
• Most mutations that delete or damage the
araC gene product shut down the araBAD
operon.
• The operon however is still inducible if a
functional araC gene is present.
• Example is that in the partial diploid araC-
araBAD+/araC+.
• This is evidence of 2 things about araC:
• 1) That araC gene product is necessary for
araBAD operon expression.
• 2) That araC acts in cis (in the same DNA
molecule) and in trans position (between
different DNA molecules.
• Some mutations in araC convert the araC
protein directly into an activator.
• araCc makes the araBAD constitutive.
• The wild type (normal araC+ allele is dominant
to araCc.
• Mutations in the araP designated araPc make
cis but not trans araBAD operons constitutive
in the absence of araC.
 Tryptophan operon
• The trp operon is required for the synthesis of
the amino acid tryptophan.
• It is a negatively controlled repressible
operon.
• It is inhibited when tryptophan (the effector
molecule) is present.
• The trpPOLEDCBA operon is negatively
regulated by a repressor.
Why is the trp operon repressible?
• Addition of tryptophan to the growth medium
causes repression of the operon.
• For this reason, the operon is said to be
repressible.
 The aporepressor
• The protein that regulates the trp operon
consists of a single structural gene called trpR
that codes for an inactive repressor protein.
• This protein is referred to as an aporepressor.
• The gene coding for the regulatory
aporepressor protein is located far from the
trp operon.
 The aporepressor
• The regulatory gene locus and the operon it
regulates are sometime said to be “unlinked”.
• This is in spite of the fact that they located on
the same single chromosome.
• When tryptophan is absent the aporepressor is
inactive and has no effect on the trp operon.
• The enzymes for the synthesis of the amino acid
are produced.
 The holorepressor
• Bacterial cells therefore make tryptophan.
• Tryptophan is a corepressor.
• This is because it acts as an effector that
converts the aporepressor into an active
repressor.
• The complex formed between tryptophan and
the aporepressor is called the holorepressor.
 The holorepressor
• When tryptophan binds to the aporepressor it
induces a conformational change.
• This converts the regulatory protein into an
active tryptophan holorepressor.
• The holorepressor binds to the trpO site and
blocks the attachment of RNA polymerase to
trpP.
• The expression of the trpPOLEDCBA operon is
shut down.
 The sequences in the trp operon
• P/O Promoter; operator sequence is found in the
promoter
• trp L: This is a leader sequence.
• 1) The leader sequence is located between the 5′
end of the trp mRNA molecule and the start
codon of the trpL gene.
• 2) It is 162 bases long and has 4 sequences called
1 and 2 and sequences 3 and 4 capable of base
pairing to form hairpin secondary structures
 2 Trp codons Stop codon
Start codon

The trpL region has 4 sequences capable of

folding into stem loops
 Leader sequence
• 3) The leader sequence also has an AUG codon
that is in-phase with a UGA stop codon.
• Together these start–stop signals encode a
polypeptide of 14 amino acids.
• 4) Two adjacent tryptophan codons are found
positions at 10 and 11 within the leader
sequence.
• The leader sequence is followed by five
structural genes.
• These code for enzymes that catalyze reactions
for the synthesis of tryptophan from chorismic
acid.
• The trp E gene for anthranilate synthetase
subunit1.
• The trp D Gene for anthranilate synthetase
subunit2.
• The trp C gene for glycerolphosphate synthetase.
 Structural genes
• The trp B gene for tryptophan synthetase
subunit1
• The trp A gene for tryptophan synthetase
subunit2.
• The operon can this be represented by
trpPOLEDCBA.
• The trp operon is regulated a mechanism
called attenuation.
 Attenuation mechanism
• An attenuator region is a hairpin structure near
the end of the leader region called trpL.
• It causes RNA polymerase to dissociate from the
DNA that it is transcribing.
• The leader region codes for an RNA that may
take different folding patterns.
• Within the leader mRNA there are four regions
capable of base pairing in various combinations
to form hairpin structures.
• A part of the leader mRNA containing regions
3 and 4 and a string of eight U's is called the
attenuator.
 Attenuation
• Attenuation depends upon the ability of
regions 1 and 2 and regions 3 and 4 of the trp
leader sequence to base pair and form hairpin
secondary structures.
Sequences 3 and 4 form a hairpin structure
that acts as a transcription termination signal.
• As soon as it forms, the RNA and the RNA
polymerase are released from the DNA.
 High tryptophan levels
• When tryptophan levels are high, the ribosome
quickly translates sequence 1 (open reading frame
encoding leader peptide).
• This blocks sequence 2 before sequence 3 is
transcribed.
• Continued transcription leads to attenuation at the
terminator-like structure formed by sequences 3 and
4.
• When tryptophan levels are low, the ribosome pauses
at the Trp codons in sequence l.
• During periods of tryptophan scarcity, a
ribosome translating the coding sequence for
the leader peptide may stall when it
encounters the two tryptophan (trp) codons.
• This is because of the shortage of tryptophan-
carrying tRNA molecules.
 Attenuation
• Because a stalled ribosome at this site blocks
region 1, a region 1+2 hairpin cannot form and an
alternative, region 2+3 hairpin is formed instead.
The region 2+3 base pairing prevents formation of
the region 3+4 transcription termination hairpin.
• Therefore RNA polymerase can move on to
transcribe the entire operon to produce enzymes
that will synthesize tryptophan.
• Formation of the paired structure between
sequences 2 and 3 prevents attenuation
because sequence 3 is no longer available to
form the attenuator structure with sequence
4.