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L12 Gene Regulation

Drosophila embryo stained for genes Fushi Tarazu


(blue) and Rhomboid (red).

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 the
time to replicate

b) Double crossover part of the fragment recombines, the cross
is 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 merozygote depending on the donar state



(b) Integrate and excise with a fragment of the neighboring
recipient DNA may carry part of the bacterial plasmid then it
is called an F plasmid and the host may be a merozygote.


(1)

Time-of-entry
mapping ( from 2)

2006 Jones and


Bartlett Publishers

L 11 Regulation of Gene Transcription:


The lac operon

The 1963 E. coli genome 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.

(3) If an episome has bacterial genes it may


integrate into the bacterial chromosome, it may
remain in the cytoplasm (below) in either
case, it may act as a partial diploid.



Otherwise, it may recombine at
homologous sequence on another plasmid
or native chromosome.

Science 28 November
2003:vol. 302 no. 5650
1488

How do you detect gene transfer?


Use restrictive media
Dominance change and colony growth
intragenic
Complementation and colony growth
intergenic
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Prokaryotic genes are (largely) up or down regulated by controlling


mRNA synthesis, activating or inducing and repressing through
protein binding to specific DNA sites.
They have to be able to recognize appropriate conditions:
external environmental conditions
metabolic requirements
infection by a virus
stress, etc.
To:
(A) turn on or (B) turn off.
In Eukaryotes, cues are also: (1) spatially significant in a cell
(2) tissue specific and
(3) developmentally specific.

Eukaryotic regulation is more complicated.

Within a cell:

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Getting a gene to express mRNA means there has to
be:

(1)Environmental conditions require specific mRNA
and in turn, protein expression.

(2) There has to be promoter that can be bound by the
RNA polymerase holoenzyme.



(3) There has to be a clear path to the start codon, a
path that is not blocked otherwise RNA polymerase will
stall and fall off the DNA template.



(4) There has to be an (unblocked) downstream
functional gene


4 kinds of transcription regulation


Default State Negative control
Positive control
Default state is on Default state is off
Regulator
Protein
Inducible
An inducer molecule
activates a gene
De-represses a gene,
turns a gene on or,
promotes transcription

Repressible A
repressor slows down
or stops the
expression of a gene

Default on, but the


system is blocked.
To express - remove
the block induce or derepress

The lac operon is the classical (first widely accepted,

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empirically sufficient) model of gene regulation.


The analysis of the lac operon was developed by
Franois Jacob and Jacques Monod in the 1950s
(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

The enzyme -galactosidase breaks down lactose,


it is only required when (environment):
(1) lactose is present,
(2) in the absence of glucose.

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Permease pumps or transports lactose into the cell


Transacetylase - not required for lactose metabolism

Regulatory Control in Jacob and Monods (1961)


operon model (in bacteria) involves:

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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.
RNA poly

repressor

binding of regulatory proteins to


particular sites sequence- on
DNA .

1+ site

promoter operator

The lac operon is a negative inducible system

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Negative control - on but


repressed
The repressor (I) binds to O
(operator site) and prevents
(most- 99.9%) transcription.
Environment and the inducer
When glucose is absent and
lactose is present (allosteric
effector), lactose (inducer) binds
to the lac repressor (allosteric
site) and inactivates its DNA
binding ability (allosteric
transition).

The lac operon is a negative inducible system

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Negative Control

Environment and the Inducer


When lactose, the inducer,
binds to the repressor,
the operon becomes induced
or de-repressed,
then transcription proceeds.

Glucose absent

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Environment - absence of glucose



Trans - acting Repressor mutants (1) If the repressor
cannot bind to the operator (I-) what will happen ?

Constitutive,

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If there is a second functional repressor (I+) on


extrachromosomal DNA, such as a plasmid or episome
an integrated fragment from an Hfr donar )

I-

mutations make a repressor that cannot bind to the


a O+ site, in this case, the operon is constitutive.
But I- mutations are recessive to I+ alleles because they
are both trans-acting.

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In the absence of glucose, and without or with (induced) lactose



+ = expression,
- = no expression

- Gal

- Gal

Genotype
Noninduced
Induced

Permease

Permease

Noninduced
Induced

Conclusion

I+ Z+Y+

-Gal & Permease


are inducible

I- Z+Y+

-Gal & Permease


are constituitive

I- Z+Y+

/ F I+

I+ is dominant to I-,
I is trans acting

and .


IS mutations 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 expression
constitutively off.
IS mutations are dominant to I+ and I- alleles.

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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.

Mutations in the repressor (I) and the three structural


genes: Z, Y, A. Non induced (- lactose), Induced (+
lactose)

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The lac operon has 3 key regulatory components


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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

Oc mutations alter the O binding site, thus the repressor cannot


bind, or the operon is constitutively on, or cannot be repressed in the
absence of lactose (and glucose).
Oc mutations are dominant to O, when both forms are present the
phenotype is Oc , i.e. there is constitutive expression of galactosidase

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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.

Many DNA binding sites have 2-fold rotational


symmetry

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Why negative control - always on

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?

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I+O+Z+Y+

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Negative control
Default state is on,

Inducible
An inducer
molecule activates
a gene
De-represses a
gene, turns a gene
on or, promotes
transcription

Repressible
A repressor slows
down or stops the
expression of a
gene

The inducer acts to remove


the repressor otherwise bound
to the operator, allowing
expression (default on)

Positive control
Default state is off-

Environment - in the presence


of glucose

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Glucose levels control the lac operon

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Figure 10-13
(1) Co- repressor (does not bind

to DNA) prevents the regulator


(cAMP) from being expressed, by
activating a repressor for the
cAMP pathway
(2) Repressor is inactivated

Cap - Camp induces high level transcription


cAMP binds to CAP

CAP-cAMP binds to the
promoter, consquently the
operon will be induced to
express at a high level

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There is a dramatic change in


the DNA double helix when
CAP- cAMP binds

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Negative control
Default state is on,

Inducible
An inducer
molecule activates a
gene
De-represses a
gene, turns a gene
on or, promotes
transcription

Repressible
A repressor slows
down or stops the
expression of a
gene

Positive control
Default state is off-

Lac operon control



The inducer (lactose) binds to
the repressor releasing it from
the operator and preventing
repressor binding, allowing
expression (on) but

A (glucose catabolite) repressor
usually inactivates (CAMP)
expression.Without CAMP, the
activator CAP does not bind to the
5 end of the promoter. Without
CAP-CAMP binding there is little
transcription (off).


Negative and positive control of the lac operon


REPRESSABLE: A
co-repressor (does
not bind to DNA)
shuts down the
regulator pathway
(cAMP) which is
necessary to activate
CAP protein

INDUCIBLE: the
inducer removes the
blocking regulator
(repressor), allowing
expression (the
default state - on) Lac
control

Positive
Default state is off

cAMP
produced,
switch on

Negative
Default state is on

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E coli

chromosome

lac operon

trp R

trp operon

Global regulation of the trp (tryptophan)


operon.

A negative repressible system.

RNA polymerase binds & transcribes

in the absence of tryptophan.



With tryptophan present, it binds to
the tryptophan repressor(trpR) which
binds to the leader sequence between
the operator and trpE - the first
structural gene

trp R

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Negative control
Default state is on,

Inducible
An inducer and /or
activator
De-represses a
gene, turns a gene
on or, promotes
transcription

Repressible
A co repressor acts
with a repressor to
slows down or stop
the expression of a
gene

Lac operon control



The inducer (lactose) binds to
the repressor releasing it from
the operator and preventing
repressor binding, allowing
expression

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

Positive control
Default state is off


A (catabolite) corepressor inhibits


CAMP expression. Without CAMP,
the CAP does not bind to the 5
end of the promoter. Without CAPCAMP binding there is little
transcription by the lac operon.

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Regulation- initiation or termination


Lactose + tryptophan: regulation of initiation ( previously
discussed).
Also regulate through termination e.g. tryptophan
attenuation in prokaryotes.

Key is
sufficient
tryptophan
available at the 2
trp codons in the
leader region (14
AA) ?

Not present-stall

Present- forms
the terminator