Transcript Section D - Prokaryotic and Eukaryotic Chromosome Structure

Section L – Regulation of
transcription in prokaryotes
Contents
L1 The lac operon
The operon, The lactose operon, The lac repressor,
Induction, cAMP receptor protein
L2 The trp operon
The tryptophan operon, The trp repressor, The
attenuator, Leader RNA structure, The leader
peptide, Attenuation, Importance of attenuation
L3 Transcriptional regulation by
alternative σ factor
Sigma factor, Promoter recognition, Heat shock,
Sporulation in Bacteriophage subtilis,
Baxteriophage σ factors
L1 The lac operon —
The operon
Operon: a unit of prokarytoic gene expression
which typically includes:
1. Structural genes for enzymes in a specific
biosynthetic pathway whose expression is coordinately controlled
2. Control elements, such as operator sequence
3. Regulator gene(s) whose products recognize the
control elements.
Control element
Structural genes
The Lac Operon
The operon, the lactose operon, the lac
repressor, induction, cAMP receptor protein
The Trp Operon
The trp operon, the trp repressor, the attenuator,
leader RNA structure, the leader peptide,
attenuation & its importance
Transcriptional regulation by alternative σ Factors
Sigma factor, promoter recognition, heat shock,
sporulation in B. subtilis, bacteriophage factors
L1 The lac operon —

The lactose operon
E. coli can use lactose as a source of carbon.
However, the enzymes required for the use of
lactose as a carbon source are only synthesized
when lactose is available as the sole carbon
source.
lacZ
lacY
lacA
codes for β-galactosidase for
lactose hydrolysis
encodes a galactoside
permease to transport
Lactose across the cell wall
encodes a thiogalactoside
transacetylase for lactose
metabolism
The lacZ, lacY, lacA genes are transcribed
from a single (lacZYA) transcription unit
under the control of a signal promoter
Plac .
LacZYA transcription unit contains an
operator site Olac
position between bases -5 and
+21 at the 3’-end of Plac
Binds with the lac repressor
L1 The lac operon —
The lac repressor
• The repressor is encoded by LacI and
active as a tetramer consisting of 4
identical subunits (has a symmetrical
structure). It binds to occupies the
operator-binding site Olac (28bp,
palindromic) and blacks almost all
transcription of lacZYA when lack of
inducer (such as lactose).
• The repressor and RNA polymerase can bind
simultaneously to the lac promoter and operator
sites. The lac repressor actually increases the
binding of the polymerase to the lac promoter by
two orders of magnitude.
• Thus, RNA polymerase binds very tightly to Plac
but no transcription occur because of the bound
repressor
L1 The lac operon —
Induction
When lac repressor binds to the inducer
(whose presence is dependent on lactose), it
changes conformation and cannot bind to
Olac site any more. This allows rapid
induction of lacZYA transcription.
Lack of inducer: the lac
repressor block all but a
very low level of transcription of lacZYA .
Absence of lactose
i
p
o
z
y
a
Active
Very low level of lac mRNA
Lactose is present, the
low basal level of
permease allows its
uptake, andβgalactosidase catalyzes
the conversion of some
lactose to allolactose.
Allolactose acts as an
inducer, binding to the
lac repressor and
inactivate it.
Presence of lactose
i
p
o
z
y
a
Inactive
Permease
Transacetylase
b-Galactosidase
• Allolactose
• causes a change in the conformation of the
repressor tetramer , reducing its affinity for the
lac operator . The lac operator is removed from
the Olac and allows the polymerase to rapidly
begin transcription of the lacZYA.
• Lactose (allolactose) is a native inducer to
release RNA transcription elongation from
Plac .
• IPTG, a synthetic inducer, can rapidly
simulate transcription of the lac operon
structural genes.
• IPTG is used to induce the expression of the
cloned gene from LacZ promoter in many
vectors, such as pUC19.
Lac promoter
MCS (Multiple cloning sites)
Ampr
pUC18
(3 kb)
lacZ’
ori
Gene X
No IPTG, little expression of X gene
With IPTG, efficient expression of X gene.
L1 The lac operon —
cAMP receptor protein

cAMP receptor protein(CRP)is a
transcriptional activator which is
activated by binding to cAMP. However,
it is only active when cAMP bound, and
cAMP is controlled by glucose. CRP
activator mediates the global regulation
of gene expression from catabolic
operons in response to glucose levels.
The Plac is a weak promoter, lacking a strong –
35 and –10 consensus sequences. High level
expression from this promoter requires the
activity of the specific activator, CRP.

When glucose is present
The level of cAMP is low in cell, and CRP
exists as a dimer which can’t bind to DNA to
regulate transcription.
When glucose is absent
The level of cAMP increase and CRP
bind to cAMP. The CRP-cAMP
complex binds to Plac just upstream
from the site for RNA polymerase.
Induces a 90°bend in DNA which
enhances RNA polymerase binding
to the promoter and thus the
transcription by 50-fold.

CRP-binding site is an inverted repeat.
Summary
C A
B
A: RNA polymerase
B: lac repressor C: CRP-cAMP
Supp. The CRP (also called CAP) protein can bind at
different sites relative to RNA polymerase.
L2 The trp operon —
The tryptophan operon
1. The trp operon encodes five structural genes required
for tryptophan synthesis.
2. It encodes a signal transcription ( 7kb, polycistron )
downstream of Otrp.
3. These genes are co-ordinately expressed when
tryptophan is in short supply in the cell.
L2 The trp operon —
The trp repressor
1. Trp repressor is encoded by a separate
operon trpR, and specifically interacts
with Otrp, a palindrome of 18 bp, and
overlaps with the Ptrp sequence between
base –21 and +3)
2. The repressor can only bind to the
operator Otrp when it is complexed with
tryptophan. Therefore, try is a corepressor and inhibits its own synthesis
through end-product inhibition (negative
feed-back regulation).
3. The repressor reduces transcription
initiation by around 70-fold, which is
much smaller than the binding of lac
repressor.
4. The repressor is a dimer of two subunits
which has a structure with a central core
and two flexible DNA-reading heads
(carboxyl-terminal of each subunit )
trpR operon
trp operon
L2 The trp operon —
free leader
RNA
The attenuator
Complementary Complementary 3:4
termination of
2:3 Elongation
of transcription transcription
L2 The trp operon —
Leader RNA structure
• The trp leader RNA contains four regions
of complementary sequence which are
capable of forming alternative hairpin
structure.
• One of these structures is the attenuator
hairpin.
L2 The trp operon —

The leader peptide
The leader RNA contains an efficient
ribosome binding site (RBS) and encodes
a 14-amino-acid leader peptide (bases 2768), Codons 10 and 11 of this peptide
encode trp. Thus the availability of trp will
affect the translation/ ribosome position,
which in turn to regulate transcription
termination.
L2 The trp operon —
Attenuation
Transcription and translation in bacteria
are coupled. Therefore, synthesis of the
leader peptide immediately follows the
transcription of leader RNA, and the
attenuation is possible
High trp
Trp is inserted at the trp
codons
Translate to the end of
leader message
Ribosome occlude
sequence 2
Terminate transcription
because 3:4 hairpin formed
Lack of trp
Lack of aminoacyl tRNAphe
Ribosome pause at trp codons ,
occluding sequence 1
2:3 hairpin (anti-terminator ) forms
Transcription into trpE
and beyond
L2 The trp operon —
Importance of attenuation
• A typical negative feed-back regulation
• Give rise to a 10-fold repression of the
trp operon transcription, increasing the
regulatory effect up to 700-fold
combining the 70-fold repressor effect.
• Faster and more subtle regulation of trp
metabolism in bacteria.
L3 Transcriptional regulation by alternative σ factor —
Sigma factor
σfactors is bifunctional protein
Bind to core RNA Pol

σ factor subunit bound to RNA
pol for transcription initiation
Released core
enzyme αββ’ω
RNA elongation
Recognize specific promoter sequence

(-35 and –10) in DNA
L3 Transcriptional regulation by alternative σ factor —
Promoter recognition
• In E.coli, σ70 is responsible for recognition
of the -10 and -35 consensus sequence.
• Differing consensus sequence are found in
sets of genes which are regulated by the
use of alternative σ factors.
L3 Transcriptional regulation by alternative σ factor —
Heat shock
The response to heat shock is one
example in E. coli where gene
expression is altered significantly by
the use of different s factors.
Heat shock
From 37ºC to
42ºC
Increase in
temperature is
more
extremely
(50ºC)
Transiently
expression of the
17 heat shock
proteins
Heat shock proteins
are the only
proteins made in E.
coli to maintain its
viability
L3 Transcriptional regulation by alternative σ factor —
Sporulation in Bacteriophage subtilis
• Under non-optimal environmental
conditions Bacillus subtilis cells from
spores through a basic cell
differentiation process involving cell
partitioning into mother cell and
forespore.
L3 Transcriptional regulation by alternative σ factor —
Baxteriophage σ factors
• Many bacteriophages synthesize their
own  factors in order to ‘take over’ the
host cell’s transcription machinery by
substituting the normal cellular  factor
and altering the promoter specificity of
the RNA polymerase.
Multiple choice questions
1. Which two of the following statements are correct?
A the double stranded DNA sequence that has the upper strand sequence 5'GGATCGATCC-3' is a palindrome.
B the double stranded DNA sequence that has the upper strand sequence 5'GGATCCTAGG-3' is apalindrome.
C the Lac repressor inhibits binding of the polymerase to the lac promoter.
D the lac operon is directly induced by lactose.
E binding of Lac repressor to allolactose reduces its affinity for the lac operator.
F IPTG is a natural inducer of the lac promoter.
2. Which one of the following statements about catabolite-regulated operons is
false?
A cAMP receptor protein (CRP) and catabolite activator protein (CAP) are different
names for the same protein.
B when glucose is present in the cell cAMP levels fall.
C CRP binds to cAMP and as a result activates transcription.
D CRP binds to DNA in the absence of cAMP.
E CRP can bend DNA, resulting in activation of transcription.
3.
A
B
C
D
E
F
4.
A
B
C
D
E
F
Which one of the following statements about the trp operon is true?
the RNA product of the trp operon is very stable.
the Trp repressor is a product of the trp operon.
the Trp repressor， like the Lac repressor, is a tetramer of identical subunits.
the Trp repressor binds to tryptophan.
tryptophan activates expression from the trp operon.
the trp operon is only regulated by the Trp represso
Which two of the following statements about attenuation at the trp operon are
true?
attenuation is rho-dependent.
deletion of the attenuator sequence results in an increase in both basal and activated
levels of tran- scription from th~ trp promoter.
the attenuator lies upstream of the trp operator sequence.
attenuation does not require tight coupling between transcription and translation.
pausing of a ribosome at two tryptophan codons in the leader peptide when
tryptophan is in short supply causes attenuation.
a hairpin structure called the pnti-terminator stops formation of the terminator hairpin,
resulting in transcriptional read-through into the trpE gene, when tryptophan is scarce.
5. Which two of the following statements about sigma factors are
false?
A the E. coli RNA polymerase core enzyme cannot start transcription
from promoters in the absence of a sigma factor subunit.
B different sigma factors may recognize different sets of promoters.
C sigma factors recognize both the -10 and -35 promoter elements.
D heat shock promoters in E. coli have different -35 and -10
sequences and bind to a diverse set of 17 heat shock sigma factors.
E sporulation in B. subtilis is regulated by a diverse set of sigma
factors.
F bacteriophage T7 expresses its own set of sigma factors as an
alternative to encoding its own RNA polymerase.
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