Transcript lac

Chapter 12
Gene Regulation
in Prokaryotes
Gene Regulation Is Necessary?
• By switching genes off when they are not needed, cells can
prevent resources from being wasted. There should be
natural selection favoring the ability to switch genes on
and off.
• Complex multicellular organisms are produced by cells that
switch genes on and off during development.
• A typical human cell normally expresses about 3% to 5% of
its genes at any given time.
• Cancer results from genes that do not turn off properly.
Cancer cells have lost their ability to regulate mitosis,
resulting in uncontrolled cell division
Classification of gene with respect
to their Expression
• Constitutive ( house keeping) genes:
– Are expressed at a fixed rate, irrespective to the cell
condition.
– They are essential for basic processes involving in cell
replication and growth
• Controllable genes:
– Are expressed only as needed. Their amount may increase
or decrease with respect to their basal level in different
condition.
– Their structure is relatively complicated with some
response elements
Regulation of gene
expression
lac operon was the first discovered example of a gene regulation
system by 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 / Medicine 1965.
•
Studied 2 different types of mutations in the lac operon:
1. Mutations in protein-coding gene sequences.
2. Mutations in regulatory sequences.
The Principles
of Transcription Regulation
• What are the regulatory proteins?
• Which steps of gene expression to be targeted?
• How to regulate? (recruitment, allostery, blocking,
action at a distance, cooperative binding)
1. Gene Expression is Controlled
by Regulatory Proteins (调控蛋白)
Gene expression is very often controlled by
Extracellular Signals, which are communicated to
genes by regulatory proteins:
 Positive regulators or activators
INCREASE the transcription
 Negative regulators or repressors
DECREASE or ELIMINATE the transcription
2. Most activators and repressors
act at the level of transcription
initiation
Why that?
1. Transcription initiation is the most energetically
efficient step to regulate. [A wise decision at
the beginning]
2. Regulation at this step is easier to do well than
regulation of the translation initiation.
Regulation also occurs at all stages after
transcription initiation. Why?
1. Allows more inputs and multiple
checkpoints.
2. The regulation at later stages allow a
quicker response.
Promoter Binding (closed
complex)
Promoter “melting” (open
complex)
Promoter escape/Initial
transcription
Elongation
Termination
3. Targeting promoter binding:
Many promoters are regulated by activators
(激活蛋白) that help RNAP bind DNA
(recruitment) and by repressors (阻遏蛋白) that
block the binding.
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Generally, RNAP binds many promoters
weakly. Why?
Activators contain two binding sites to bind
a DNA sequence and RNAP simultaneously,
can therefore enhance the RNAP affinity with
the promoters and increases gene
transcription. This is called recruitment
regulation (招募调控).***
On the contrary, Repressors can bind to the
operator inside of the promoter region, which
prevents RNAP binding and the transcription
of the target gene.
a. Absence of
Regulatory Proteins:
basal level
expression
b. Repressor
binding to the
operator represses
expression
c. Activator
binding activates
expression
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4 Targeting transition to the open
complex: Allostery regulation (异构调控)
after the RNA Polymerase Binding
In some cases, RNAP binds the promoters
efficiently, but no spontaneous isomerization (异构化)
occurs to lead to the open complex, resulting in no or low
transcription.
Some activators can bind to the closed complex,
inducing conformational change in either RNAP or DNA
promoter, which converts the closed complex to open
complex and thus promotes the transcription. This is an
example of allostery regulation.
Allostery regulation
Allostery is not only a mechanism of gene activation , it is
also often the way that regulators are controlled by their
specific signals.
Repressors can work in ways:
(1) blocking the promoter binding.
(2) blocking the transition to the
open complex.
5. Action at a Distance and DNA Looping. The
regulator proteins can function even binding at a
DNA site far away from the promoter region,
through protein-protein interaction and DNA
looping.
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DNA-binding protein can facilitate interaction
between DNA-binding proteins at a distance
Architectural protein
6. Cooperative binding (recruitment)
and allostery have many roles in
gene regulation
For example: group of regulators often bind DNA
cooperatively (activators and/or repressors interact
with each other and with the DNA, helping each
other to bind near a gene they regulated) :
(1) produce sensitive switches to rapidly turn on a gene
expression. (1+1>2)
(2) integrate signals (some genes are activated when
multiple signals are present).
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Topic 2: Regulation of
Transcription Initiation :
Examples from Bacteria
OPERON in gene regulation of
prokaryotes
• Definition: a cluster of genes in which expression is regulated by
operator-repressor protein interactions, operator region, and the
promoter.
• Its structure: Each Operon is consisted of few structural
genes( cistrons) and some cis-acting element such as promoter (P)
and operator (O).
• Its regulation: There are one or more regulatory gene outside of the
Operon that produce trans-acting factors such as repressor or
activators.
• Classification:
•
1- Catabolic (inducible) such as Lac OPERON
2- Anabolic (repressible) such as ara OPERON
•
3- Other types
General structure of an OPERON
First example:
Lac operon
The lactose Operon
(乳糖操纵子)
Point 1: Composition of
the Lac operon
1. Lactose operon contains 3 structural
genes and 2 control elements.
The enzymes encoded by lacZ, lacY, lacA are
required for the use of lactose as a carbon source.
These genes are only transcribed at a high level
when lactose is available as the sole carbon source.
The LAC operon
lacZ
codes for β-galactosidase (半乳
糖苷酶) for lactose hydrolysis
lacY
encodes a cell membrane protein
called lactose permease (半乳糖苷
渗透酶) to transport Lactose
across the cell wall
lacA encodes a thiogalactoside
transacetylase (硫代半乳糖苷转乙
酰酶)to get rid of the toxic
thiogalacosides
The lacZ, lacY, lacA genes are transcribed into a
single lacZYA mRNA (polycistronic mRNA) under
the control of a single 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
Control elements
Regulatory Gene
Operon
i
Plac Olac
z
y
a
DNA
-5 +21
m-RNA
Protein
repressor
β-Galactosidase
Transacetylase
Permease
Point 2: Regulatory proteins
and their response to
extracellular signals
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2. An activator and a repressor
together control the Lac operon
expression
The activator: CAP (Catabolite Activator Protein,
代谢产物激活蛋白) or CRP (cAMP Receptor
Protein,cAMP受体蛋白); responses to the glucose
level.
The repressor: lac repressor that is encoded by
LacI gene; responses to the lactose.
Sugar switch-off mechanism
The LAC operon
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3. The activity of Lac repressor and
CAP are controlled allosterically by
their signals.
Allolactose binding: turn off Lac repressor
cAMP binding: turn on CAP
Lactose is converted to allolactose by b-galactosidase,
therefore lactose can indirectly turn off the repressor.
Glucose lowers the cellular cAMP level, therefore,
glucose indirectly turn off CAP.
The LAC operon
Lac OPERON an inducible Operon
In the absence
of lac
In the presence
of lac
CRP or CAP is positive regulator of Lac and
some other catabolic Operons
CRP= Catabolic gene regulatory Protein
CRP= cAMP receptor Protein
CAP= Catabolic gene Activating Protein
Regulation of lac Operon Expression
Off
Off
Functional state of the E. coli lac operon
in the absence of lactose:
Functional state of the E. coli lac operon
growing on lactose:
Positive control of
the lac operon
with CAP
Point 3: The mechanism
of the binding of
regulatory proteins to
their sites
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4. CAP and Lac repressor have
opposing effects on RNA polymerase
binding to the promoter
Repressor binding physically
prevents RNAP from binding to the
promoter, because the site bound
by lac repressor is called the lac
operator (Olac ), and the Olac
overlaps promoter (Plac).
The LAC operon
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CAP binds to a site upstream of the
promoter, and helps RNA
polymerase binds to the promoter
by physically interacting with RNAP.
This cooperative binding stabilizes
the binding of polymerase to Plac.
Base pair sequence of controlling sites, promoter, and operator for
lac operon of E. coli.
5. CAP interacts with the CTD
domain of the a-subunit of RNAP
• CAP interacts with the CTD domain of the a-subunit of
RNAP and thus promotes the promoter binding by
RNAP
a CTD: C-terminal domain of the a subunit of RNAP
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Lactose/allolactose is a native inducer to
release RNA transcription from Plac.
IPTG (isopropyl--D-thiogalactopyranoside,异丙基-β-D-硫代吡喃半乳糖
苷 ), a synthetic inducer, can rapidly
stimulate transcription of the lac operon
structural genes.  IPTG is used to
induce the expression of the cloned gene
from lac promoter in many vectors, such
as pUC19.
Lac promoter
MCS (Multiple cloning sites,
多克隆位点）
Ampr
pUC18
(3 kb)
lacZ’
ori
Gene X
No IPTG, little protein X
With IPTG, a lot of protein X
Back
Second example:
The Trp operon of E. coli
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Trp OPERON a repressible example
In the absence of Trp
In the presence of Trp
Regulation of the trp operon:
1. Repressor/operator interaction
– When tryptophan is present, tryptophan binds to trpR
gene product.
– trpR protein binds to the trp operator and can only bind
to the operator, thus prevents transcription.
– Repression reduces transcription of the trp operon ~70fold.
2. Molecular model for attenuation(弱化作用）:
•
Recall that a leader region (trpL) occurs between the
operator and the trpE sequence.
•
Within this leader is the attenuator sequence (att).
•
att sequence contains a start codon, 2 Trp codons, a stop
codon, and four regions of sequence that can form three
alternative secondary structures.
Secondary structure
Signal
•
Paired region 1-2
pause
•
Paired region 2-3
anti-termination
•
Paired region 3-4
termination
Organization of the leader/attenuator trp operon sequence.
Attenuation model in Trp starved cells
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):
1.
Trp-tRNAs are unavailable, ribosome stalls at Trp codons and
covers attenuator region 1.
2.
Region 1 cannot pair with region 2, instead region 2 pairs
with region 3 when it is synthesized.
3.
Region 3 (now paired with region 2) is unable to pair with
region 4 when it is synthesized.
4.
RNA polymerase continues transcribing region 4 and beyond
synthesizing a complete trp mRNA.
Attenuation model in Trp non-starved cells
Molecular model for attenuation (cont.):
Position of the ribosome plays an important role in attenuation:
When Trp is abundant (and not required):
1.
Ribosome does not stall at the Trp codons and continues
translating the leader polypeptide, ending in region2.
2.
Region 2 cannot pair with region 3, instead region 3 pairs
with region 4.
3.
Pairing of region 3 and 4 is the “attenuator” sequence and
acts as a termination signal.
4.
Transcription terminates before the trp synthesizing genes
are reached.
The attenuators of some operons
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