Upper Strand Sequences of E.coli promoters

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Bacterial Gene Regulation II
Dr. David S. Gross Office 7-215A Phone
675-5027 Email dgross_at_lsuhsc.edu
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lac operon
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Lactose metabolism in E.coli hinges on the
presence of two proteins
Galactoside Permease lacY gene product b-Galactos
idase lacZ gene product
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whose expression is under control of the lac
operon
galactoside permease
b-galactosidase
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IPTG is a non-metabolizable inducer of the lac
operon
it regulates the DNA binding activity of a
sequence-specific protein, Lac repressor.
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Induction profile of b-galactosidase in E. coli
IPTG removed
IPTG added
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IPTG induces a dramatic conformational change in
Lac Repressor, product of the lacI gene
IPTG
(dark shading)
DNA binding domains are stable only in DNA
bound state (-IPTG).
Lac repressor tetramer of identical monomers

-IPTG
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b-gal is initially synthesized in the
repressor-free environment of I- Z- cells
following transfer of I Z DNA
(IPTG)

IPTG

following transfer of F factor
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The lac operon
Transcription start site
Transcription termination site
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LacO3
LacO2
LacO1
ATG
TAA
ATG
TAA
TAA
ATG
promoter
lacZ
lacY
lacA
polycistronic mRNA transcript
Shine Dalgarno sequence
Operon One promoter, one transcriptional stop
multiple cistrons (genes), each with its own
translational start and stop.
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Lac Repressor tightly binds lac O1, the principal
lac operator
5-
-3
(lac O1)
lac O1 is palindromic lower strand sequence
5
3
AATTGTTATCCGCTCACAATT
Repressor contacts precisely the 16/21 bases that
comprise the palindromic sequence (red)
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Lac repressor binds cooperatively to two DNA
elements the main Operator O1 and a pseudo
Operator (O2 or O3)
Pseudo Operator O2
Pseudo Operator O3
lac ZYA operon
lac I gene
Main Operator
92 bp 400 bp

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• Cooperativity is defined as "the interaction
  between either identical or different binding
  sites of a macromolecule so that the binding of a
  ligand to one site affects the binding of
  subsequent ligands to other sites on the
  molecule."
• Macromolecule Lac Repressor
• (tetramer functions as two functionally
  distinct dimers)
• Ligand DNA (operator sites 1, 2 and 3)

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Cooperative binding of Lac Repressor results in
the formation of a DNA Loop
Lac Repressor tetramer
O1
O2

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SummaryRole ofLac Repressor
(e.g., lactose, IPTG)
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Even when lactose is present, and lacI has
dissociated from the operator, the lac operon
may not be fully active.
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Positive Regulation of the lac Operon requires
CAP
no binding
O
P
no binding
(1X)
due to Catabolite Repression
CAP catabolite activator protein aka CRP
cAMP receptor protein

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Positive Regulation of the lac Operon requires CAP
O
P
no binding
CAP
(50X)
lactose-bound lac repressor
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RNAP and cAMP-CAP bind cooperatively to the lac
promoter
Regulatory sequences

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Catabolite Repression
Phenomenon in which glucose, E. colis
preferred catabolite, diminishes expression of
genes specifying proteins involved in the
fermentation of other sugars.
How does it work?
BY PREVENTING CAP FROM BINDING TO ACTIVATOR SITES
upstream of weak promoters (containing weak 35
and 10 regions), such as those regulating
operons which catabolize sugars such as lactose,
arabinose and galactose. In order for RNAP to
bind such promoters, it requires help from
CAP. CAP can only bind DNA as a cAMP-CAP complex.

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Catabolite Repression lac mRNA levels are
drastically inhibited by glucose
Glucose
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Catabolite Repression
Ultimately stems from a drop in intracellular
cAMP level
How are cAMP levels regulated?
Intracellular concentrations of cAMP are greatly
reduced in the presence of glucose. Adenylate
cyclase, responsible for the production of cAMP,
is activated by a phosphorylated enzyme, EIII
. EIII is itself dephosphorylated
upon transport of glucose through the cell
membrane. Glucose also stimulates cAMP
transport out of the cell.
glc
glc

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Catabolite Repression rapid but not immediate
Glucose
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• Glucose does not affect the abundance of lac mRNA
  immediately because
• 1. synthesis of cAMP must cease
• 2. intracellular cAMP levels must drop (actively
  transported out of cell)
• 3. cAMP must dissociate from CAP
• 4. CAP must release from lac promoter DNA
• 5. recruitment of RNAP, and concomitant
  transcription initiation, must decline
• 6. pre-synthesized lac mRNA must be degraded by
  intracellular RNases (responsible for the short
  half life of virtually all bacterial mRNAs)

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Catabolite Repression
IS NOT AN EXAMPLE OF NEGATIVE CONTROL
Rather, catabolite repression occurs since the
stimulatory effect of CAP is lost (for it is
unable to bind DNA).
CATABOLITE REPRESSION RESULTS FROM A LOSS OF
POSITIVE CONTROL.
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Summary Role of glucose and lactose in
regulating transcription of the lac operon
relative transcription level
1.0

0.04
-
cAMP
50
-
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lac operon summary
Subject to both positive and negative regulation
1 Negatively regulated (repressed) by lac
repressor under conditions of lactose
deprivation. 2 Positively regulated
(activated) by CAP/CRP under conditions of
glucose deprivation. Together, these two
mechanisms result in a 1300-fold range of
expression.
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ara operon
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The araC and araBAD operons
Exhibit both negative and positive control
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Mechanism of araBAD regulation
Repression at a distance mediated by DNA looping
(glucose high)
...as well as its own (araC) transcription.

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Positive control mediated by arabinose
(no transcription)
(c) When arabinose is abundant, it binds AraC,
inducing a conformational change and converting
AraC to an activator. The DNA loop is opened, and
AraC binds to each half-site of araI (AraC
remains bound to araO1). If glucose is also low
(cAMP high), CAP-cAMP binds the CAP site and
activates araBAD transcription.
Note AraC still acts as a repressor of the araC
gene even though bound by its ligand.

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araBAD operon summary
Subject to both positive and negative regulation
1 Negatively regulated by araC under
conditions of arabinose deprivation. 2
Positively regulated by CAP/CRP under conditions
of glucose deprivation. 3 Positively
regulated by araC under conditions of arabinose
abundance.
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trp operon
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trp operon
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The trp operon is negatively regulated by the
Trp repressor
Regulatory region
Trp repressor binds its target DNA sequence only
when it itself is bound by its corepressor,
tryptophan.
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The trp operator is a palindromicDNA sequence
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trp repressor dimer binds its operator in
successive major grooves of the DNA

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Tryptophan induces a conformational change in Trp
Repressor

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Expression of the trp operonis fine-tuned by a
second mechanism (transcription attenuation) that
relies on the close coupling of transcription and
translation in E. coli
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Transcriptional attenuation of the trp operon is
regulated by abundance of charged tRNATrp
(Attenuator)
mRNA
Ribosome translating
DNA
trpE
mRNA

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compare attenuator
(Attenuator)
mRNA
UUUUUUU
Ribosome translating trpL
DNA
to a typical transcription terminator
mRNA
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Transcriptional attenuation of the trp operon is
regulated by abundance of charged tRNATrp
Attenuator
mRNA
Ribosome translating
DNA
trpE
mRNA

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Terminator and anti-terminator conformations of
the trp leader (trpL) sequence
2-3
1-2
3-4
Anti-Terminator
Terminator
Prevents formation of 3-4 stem loop
GC-rich stem followed by a series of U residues
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trp operon summary
Two negative regulatory mechanisms 1 trp
repressor binding to trpO operator, thereby
sterically occluding the trp promoter
(trpP). 2 ribosome facilitating formation of
the terminator (attenuator) stem-loop structure
within the 5-end of the trp mRNA (by occupying
segment 2 within trpL). contribute to the
700-fold dynamic range of transcription.