l 12 Bio l 2612013 p Regulation

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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

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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

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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.

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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

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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

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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

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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

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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.

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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

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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 )

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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.

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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

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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.

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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.

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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

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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.

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Many DNA binding sites have 2-fold rotational

symmetry


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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?

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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

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Environment - in the presence

of glucose

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Glucose levels control the lacoperon

Figure 10-13

Cap - Camp induces high level transcription

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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

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Negativecontrol

Default state is on, Positivecontrol


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).

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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

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cAMP

produced,switch on


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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

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trp R


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Negative control

Default state is on,

Positive control


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.

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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

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