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L12 Gene Regulation
Drosophila embryo stained for genes Fushi Tarazu(blue) and Rhomboid(red).
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1) F+ plasmid converts an F- cell to an F+ - could be an episome
2) A partial Hfr foreign chromosome , a fragment crosses from an
a Hfr cell to a recipient cell.
a) Single crossover no recombination, fragment degraded in thetime to replicateb) Double crossover part of the fragment recombines, the crossis not reciprocal and the recipient (host) is recombinant, whereas
the recombinant fragment is degraded in the time to replicate
(3) Whole Hfr plasmid crosses intact.(a)Not integrated or integrates and cleanly excise possibly a
partial diploid merozygotedepending on the donar state
(b) Integrate and excise with a fragment of the neighboringrecipient DNA may carry part of the bacterial plasmid then itis called an F plasmid and the host may be a merozygote.
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2006 Jones and
Bartlett Publishers
Time-of-entry
mapping ( from 2)
3
(1)
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L 11 Regulationof Gene Transcription:The lac operon
4
The 1963E. coligenome map,
constructed by timing the transfer
of a sequence of genes starting at
the origin (0 minutes), and the
time genes on one strand of DNA
take to get through a congugation
tube into a second host bacteria.
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How do you detect gene transfer?Use restrictive media
Dominance change and colony growth
intragenic
Complementation and colony growth
intergenic
6
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Prokaryotic genes are (largely) up or downregulated by controlling
mRNA synthesis, activating or inducingand repressingthrough
protein binding to specific DNA sites.
They have to be able to recognize appropriate conditions:
external environmental conditions
metabolic requirementsinfection by a virus
stress, etc.
To: (A) turn onor (B)turn off.
In Eukaryotes, cues are also:(1) spatially significantin a cell(2) tissue specificand
(3) developmentally specific.
Eukaryotic regulation is more complicated.
7
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Within a cell:Getting a gene to express mRNA means there has to
be:(1)
Environmental conditions require specific mRNAand in turn, protein expression.
(2) There has to be promoterthat can be bound by theRNA polymerase holoenzyme.
(3) There has to be a clear path to the start codon, apath that is not blocked otherwise RNA polymerase willstall and fall off the DNA template.
(4) There has to be an (unblocked) downstreamfunctional gene
8
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4 kinds of transcription regulation
Default State
Regulator
Protein
Negative control
Default state is on
Positive controlDefault state is off
InducibleAninducermoleculeactivates a gene
De-represses a gene,
turns a gene on or,promotes transcription
Default on, but the
system is blocked.
To express - remove
the block induce or de-repress
RepressibleArepressor slows down
or stops theexpression of a gene
9
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The lac operonis the classical (first widely accepted, 10
empirically sufficient) model of gene regulation.
The analysis of the lac operon was developed by
Franois Jacob and Jacques Monod in the 1950
s
(Nobel Prize laureates).
The lac operon contains three co-linear structural genes:
Z galactosidase an enzyme that cleaves lactose
into glucose and galactose
Y- lactose permease facilitates lactose uptake
into the cell
A transacetylase - is unrelated to lactose metabolism
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The enzyme -galactosidase breaks down lactose,
it is only required when (environment):
(1)lactose is present,(2)in the absence of glucose.
Permease pumps or transports lactose into the cell
Transacetylase - not required for lactose metabolism
11
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binding of regulatory proteins to
particular sites sequence- on
DNA .
Regulatory Control in Jacob and Monods (1961)
operon model(in bacteria) involves:
There is a region between the promoter and the 1+ base (cis),
that can bind a repressor which diffuses to the site - trans acting.
The bound repressor protein is large enough to cover the
operator, overlap the promoter and the 1+ base.
promoter operator
RNA polyrepressor
12
1+ site
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The lac operon is a negative induciblesystem
Environment and theinducer
When glucose is absent and
lactose is present (allosteric
effector), lactose (inducer)binds
to the lac repressor (allosteric
site) and inactivates its DNAbinding ability (allosteric
transition).
Negativecontrol - on but
repressedThe repressor (I) binds to O
(operator site) and prevents
(most- 99.9%) transcription.
13
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The lac operon is a negative induciblesystem
Environment and the Inducer
When lactose, the inducer,binds to the repressor,
the operon becomes induced
or de-repressed,
thentranscription proceeds.
Negative Control
14
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15Glucose absent
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Trans - actingRepressor mutants (1) If the repressorcannot bind to the operator (I-) what will happen ?
Constitutive,
Environment - absence of glucose16
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If there is a second functional repressor (I+) on
extrachromosomal DNA, such as a plasmid or episomean integrated fragment from an Hfr donar )
17
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I-mutations make a repressor that cannot bind to the
a O+ site, in this case, the operon isconstitutive.
But I-mutations are recessive to I+alleles because they
are both trans-acting.
18
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!- Gal !- Gal Permease Permease
Genotype Noninduced Induced Noninduced Induced Conclusion
I+Z+Y+ - + - + !-Gal & Permeaseare inducible
I-Z+Y+ + + + + !-Gal & Permeaseare constituitive
I-Z+Y+
/ F I+
- + - + I+is dominantto I-,
I is trans acting
and .
In the absence of glucose, and without or with (induced) lactose
+ = expression, - = no expression
19
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ISmutations make a super-repressor- it binds the DNA of the O
site, but this repressor cannot bind lactose, it cannot be
derepressed. The repressor - operon block, renders expressionconstitutively off.
ISmutations are dominant to I+ and I- alleles.
20
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21
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Cis - acting mutations on the same strand
Each gene may or may not have a
loss-of- function mutation (Z-, Y-, A- ),
that produces a non functional transcript or protein.
Z-, Y-and A-loss-of -function mutations on the
bacterial chromosome are recessive, one functional
copy on a plasmid is sufficient for expression.
22
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The lac operon has 3 key regulatory components
trans- lac repressor a protein from the lac I gene
cis- lac operator site (O) DNA binding site of repressor
cis- lac promoter (P) DNA binding site of RNA polymerase
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Ocmutations alter the O binding site, thusthe repressor cannot
bind, orthe operon is constitutively on, or cannot be repressed in the
absence of lactose (and glucose).
Ocmutations are dominant to O, when both forms are present thephenotype is Oc, i.e. there is constitutive expression of galactosidase
25
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The lac operator region.Mutations affect the base sequence
including the Operator site, affecting how well the repressor
binds.
Oc(constitutive) mutations prevent the repressor binding.
26
Many DNA binding sites have 2-fold rotational
symmetry
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27
Why negative control - always on
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The Lac promoter element. P-mutations affect the base
sequence in Promoter region, affecting how well RNA
polymerase binds. If RNA polymerase does not bind - will
there be transcription?
28
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29I+O+Z+Y+
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Negative control
Default state is on,
Positive control
Default state is off-
InducibleAninducer
molecule activates
a gene
De-represses agene, turns a gene
on or, promotestranscription
The inducer acts to remove
the repressor otherwise bound
to the operator, allowing
expression(default on)
Repressible
A repressor slowsdown or stops theexpression of a
gene
30
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Environment - in the presence
of glucose
31
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Glucose levels control the lacoperon
Figure 10-13
Cap - Camp induces high level transcription
32
CAP-cAMP binds to the
promoter, consquently the
operon will be induced to
express at a high level
(1) Co- repressor (does not bind
to DNA) prevents the regulator
(cAMP) from being expressed, by
activating a repressorfor the
cAMP pathway
(2) Repressor is inactivated
cAMP binds to CAP
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There is a dramatic change inthe DNA double helix when
CAP- cAMP binds
33
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Negativecontrol
Default state is on, Positivecontrol
Default state is off-
InducibleAninducer
molecule activates a
geneDe-represses a
gene, turns a gene
on or, promotestranscription
Lac operon control
The inducer (lactose) binds to
the repressor releasing it from
the operator and preventing
repressor binding, allowing
expression (on) but
RepressibleA repressor slows
down or stops theexpression of a
gene
A (glucose catabolite) repressor
usually inactivates (CAMP)expression.Without CAMP, the
activator CAP does not bind to the
5end of the promoter. Without
CAP-CAMP binding there is little
transcription (off).
34
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Negative and positive control of the lacoperon
PositiveDefault state is off
REPRESSABLE: A
co-repressor (doesnot bind to DNA)shuts down the
regulator pathway
(cAMP) which isnecessary to activate
CAP protein
NegativeDefault state is on
INDUCIBLE: theinducer removes the
blocking regulator
(repressor), allowingexpression (thedefault state - on) Lac
control
35
cAMP
produced,switch on
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36
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trp operon
lac operon
trp R
E colichromosome
Global regulation of the trp (tryptophan)operon.A negative repressible system.
RNA polymerase binds & transcribesin the absence of tryptophan.With tryptophan present, it binds tothe tryptophan repressor(trpR) whichbinds to the leader sequence between
the operator and trpE - the firststructural gene
37
trp R
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Negative control
Default state is on,
Positive control
Default state is off-
InducibleAninducerand /or
activator
De-represses a
gene, turns a geneon or, promotes
transcription
Lac operon control
The inducer (lactose) binds to
the repressor releasing it from
the operator and preventing
repressor binding, allowing
expression
RepressibleA co repressor acts
with a repressor toslows down or stop
the expression of agene
Tryptophan global regulation
A repressor is produced by the
trp R site, but it cannot bind
to the tryptophan operator
unless tryptophan first binds
to it (corepressor), together
the tryptophan /repressor
down-regulates the
expression of tryptophan
A (catabolite) corepressor inhibits
CAMP expression.Without CAMP,
the CAP does not bind to the 5
end of the promoter. Without CAP-
CAMP binding there is little
transcription by the lac operon.
38
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Regulation- initiation or termination
Lactose + tryptophan: regulation of initiation ( previouslydiscussed).
Also regulate through termination e.g. tryptophan
attenuation in prokaryotes.
Key issufficienttryptophanavailable at the 2trp codons in the
leader region (14AA) ?Not present-stallPresent- formsthe terminator