L1 The lac operon

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Molecular Biology

• L1 The lac operon
• L2 The trp operon
• L3 Transcriptional regulation by
• alternative s factors

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Molecular Biology
L1The LAC Operon

• Operon - what is it?
• The operon is a unit of gene expression and
  regulation
• The structural genes (any gene other than a
  regulator) for enzymes involved in a specific
  biosynthetic pathway whose expression is
  co-ordinately controlled.
• Control elements such as an operator sequence,
  which is a DNA sequence that regulates
  transcription of the structural genes.
• Regulator gene(s) whose products recognize the
  control elements, for example a repressor which
  binds to and regulates an operator sequence.

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Molecular Biology
The structure of operon
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Molecular Biology
Francois Jacob and Jacques Monod

• (Pasteur Institute, Paris, France)
• Studied the organization and control of the lac
  operon in E. coli.
• Earned Nobel Prize in Physiology or Medicine
  1965.

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Molecular Biology
E. colis lac operon

• E. coli expresses genes for glucose metabolism
  continuously.
• Metabolism of other alternative types of sugars
  (e.g., lactose) are regulated specifically.
• Lactose disaccharide (glucose galactose),
  provides energy.
• Lactose acts as an inducer (effector molecule)
  and stimulates expression of three proteins at
  1000-fold increase
• ?-galactosidase (lacZ)
• An enzyme responsible for hydrolysis of
  lactose to galactose and glucose .
• Permease (lacY)
• An enzyme responsible for lactose transport
  across the bacterial cell wall.
• Acetylase (lacA) Function is not understood.

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Molecular Biology
?-galactosidase and structure of lactose
E. Coli cells need an enzyme to break the lactose
down into its two component sugars galactose and
glucose. The enzyme that cuts it in half is
called ? -galactosidase.
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Molecular Biology
Structure of the lac operon
Lac operon
Acetylase
?-galactosidase
Permease
DNA
lacI promoter-lacI-terminator operon promote
r-operator-lacZ-lacY-lacA-terminator
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Molecular Biology
Without inducer-no structure genes expression
Lac operon
Regulation genes
?-galactosidase
Permease
Acetylase
DNA
transcript
No structure genes expression
mRNA of repressor
translate
Inactive lac repressor
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Molecular Biology
Binding of inducer inactivates the lac repressor
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Molecular Biology
cAMP receptor protein
The Plac promoter is not a strong promoter. Plac
and related promoters do not have strong -35
sequences and some even have weak -10 consensus
sequences. For high level transcription, they
require the activity of a specific activator
protein called cAMP receptor protein (CRP). CRP
may also be called catabolite activator protein
or CAP. Glucose reduces the level of cAMP in the
cell. When glucose is absent, the levels of cAMP
in E. coli increase and CRP binds to cAMP.So, the
CRP-cAMP complex binds to the lactose operon.
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Molecular Biology
DNA-bending and Transcription regulation
CAP-cAMP binding to the lac activator-binding
site recruits RNA polymerase to the adjacent lac
promoter to form a closed promoter complex. This
closed complex then converts to an open promoter
complex. CAP-cAMP bends its target DNA by about
90 when it binds. And this is believed to
enhance RNA polymerase binding to the promoter,
enhancing transcription by 50-fold.
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Molecular Biology
L2 The TRP operon

• If amino acids are present in the growth medium
  E. coli will import amino acids before it makes
  them, genes for amino acid synthesis are
  repressed.
• When amino acids are absent in the growth medium,
  genes are turned on (or expressed) and amino
  acid synthesis occurs.
• The tryptophan (Trp) operon of E. coli is one of
  the most extensively studied operons in amino
  acids synthesis.
• first characterized by Charles Yanofsky et al.

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Molecular Biology
Structure of the trp operon and function of the
trp repressor
A gene product of the separate trpR operon, the
trp repressor, specifically interacts with the
operator site of the trp opseron. The symmetrical
operator sequence, which forms the trp
repressor-binding site, overlaps with the trp
promoter sequence between bases -12 and 3.
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Molecular Biology
The attenuator
At first, it was thought that the repressor was
responsible for all of the transcriptional
regulation of the trp operon. However, it was
observed that the deletion of a sequence between
the operator and the trpE gene coding region
resulted in an increase in both the basal and the
activated level if transcriptio. This site is
termed the attenuator and it lies towards the end
of transcribed leader sequence of 162 nt that
precedes the trpE initiator codon. The attenuator
is a rho-independent terminator site which has a
short GC-rich palindrome followed by eight
successive U residues. If this sequence is able
to form a hairpin structure in the RNA
transcript, then it acts as highly efficient
transcription terminator and only a 140bp
transcript is synthesized.
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Molecular Biology
Leader RNA structure and leader peptide
14aa
hairpins
Pause
Anti-termination
Termination
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Molecular Biology
Molecular model for attenuation (cont.)

• Position of the ribosome plays an important role
  in attenuation
• When Trp is scarce or in short supply (and
  required)
• Trp-tRNAs are unavailable, ribosome stalls at Trp
  codons and covers attenuator region 1.
• Region 1 cannot pair with region 2, instead
  region 2 pairs with region 3 when it is
  synthesized.
• Region 3 (now paired with region 2) is unable to
  pair with region 4 when it is synthesized.
• RNA polymerase continues transcribing region 4
  and beyond synthesizing a complete trp mRNA.

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Molecular Biology
Molecular model for attenuation (cont.)

• Position of the ribosome plays an important role
  in attenuation
• When Trp is abundant (and not required)
• Ribosome does not stall at the Trp codons and
  continues translating the leader polypeptide,
  ending in region2.
• Region 2 cannot pair with region 3, instead
  region 3 pairs with region 4.
• Pairing of region 3 and 4 is the attenuator
  sequence and acts as a termination signal.
• Transcription terminates before the trp
  synthesizing genes are reached.

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Molecular Biology
Importance of attenuation

• The presence of tryptophan gives rise to a
  10-fold repression of trp operon transcription
  through the process of attenuation alone.
• Combined with control by the trp repressor
  (70-fold), thus means that tryptophan levels
  exert a 700-fold regulatory effect on expression
  from the trp operon.
• Attenuation occurs in at least six operons that
  encode enzyme concerned with amino acid
  biosynthesis.

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Molecular Biology
L3 Transcriptional regulation by
alternativesfactors

• s factors appear to be bifunctional proteins that
  stimultaneously can bind to core RNA polymerase
  and recognize specific promoter sequence in DNA.
• Many bacteria, including E. coli, produce a set
  ofsfactors that recognize different sets of
  promoters.
• Some environmental conditions require a massive
  change in the overall pattern of gene expression
  in the cell.
• Under such circumstances, bacteria may use a
  different set of s factors to direct RNA
  polymerase binding to different promoter
  sequences.

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Molecular Biology
Promoter recognition
The binding of an alternative sfactors to RNA
polymerase can confer a new promoter specificity
on the enzyme responsible for the general RNA
synthesis of the cell. Comparisons of promoters
activated by polymerase complexed to specific
sfactors show that each sfactor recognizes a
different combination of sequences centered
approximately around the -35 and -10 sites. It
seems likely that sfactors themselves contacet
both of these regions, with the -10 region being
most important. The s70 subnuit is the most
common sfactor in E. coli which is responsible
for recognition of general promoters which have
consensus -35 and -10 elements.
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Molecular Biology
Heat shock promoter
Comparison of the heat-shock (s32) and general
(s70) responsive promoters
When E. coli is subjected to an increase in
temperature, the synthesis of a set of around 17
proteins, called heat-shock proteins, is induced.
The promoters for E. coli heat-shock
proteins-encoding genes are recognized by a
unique form of RNA polymerase holoenzyme
containing a variant sfactor s32, which is
encoded by the rpoH gene.
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Molecular Biology
Sporulation in Bacillus subtilis
Vegetatively growing B.subtilis cells from
bacterial spores(see Topics A1) in response to a
sub-optimal environment.The RNA polymerase in
B.subtilis is functionally identical to that in
E.coli. The vegetatively growing B.subtilis
contains a diverse set of sfactors. Sporulation
is regulated by a further set of sfactors in
addition to those of the vegetative cells.
Different sfactors are specifically active before
cell partition occurs, in the forespore and in
the mother cell. Cross-regulation of this
compartmentalization permits the forespore and
mother cell to tightly co-ordinate the
differentiation process.
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Molecular Biology
Bacteriophage sfactors
Some bacteriophages provide new ssubunits to
endow the host RNA polymerase with a different
promoter specificity and hence to selectively
express their own phage genes(e.g. phage T4 in
E.coli and SPO1 in B.subtilis). This stragety is
an effective alternative to the need forfor the
phage to encode its own complete polymerase(e.g.
bacteriophage T7,see Topic K2). The B.subtilis
bacteriophage SPO1 expresses a cascade of
sfactors in sequence to allow its own genes to be
transcribed at specific stage during virus
infection. Initially, early genes are expressed
by normal bacterial holoenzyme. Among these early
genes is the gene encoding s28, which then
displaces the bacterial sfactor from the RNA
polymerase. The s28-containing holoenzyme is then
responsible for expression of the middle genes.
The phage middle genes include genes 33 and 34
which specificy a further s factor that is
responsible for the specific trancription of late
genes. In this way, the bacteriophage uses the
hosts RNA polymerase machinery and expresses its
genes in a defined sequential order.