lect3 transcriptional control

8/3/2019 Lect3 Transcriptional Control

1/23

TranscriptionalTranscriptional

controlcontrol


2/23

Coordinately regulatedCoordinately regulated

genes are not linked ingenes are not linked in

eukaryoteseukaryotes In prokaryotes, several genes encoding forIn prokaryotes, several genes encoding forparticular proteins will link together in an operon.particular proteins will link together in an operon.

When response to the activating signal, the geneWhen response to the activating signal, the gene

will be transcribed as one single polycistronicwill be transcribed as one single polycistronicmRNA.mRNA.

Translation of the mRNA will result into individualTranslation of the mRNA will result into individual

proteins following binding of the ribosome atproteins following binding of the ribosome atparticular sites.particular sites.

Eg: lac operon where three genes encoding forEg: lac operon where three genes encoding forthree different proteins are linked together and arethree different proteins are linked together and areinduced in the presence of lactoseinduced in the presence of lactose


3/23

In eukaryotic organisms , this type ofIn eukaryotic organisms , this type of

linkage does not occur.linkage does not occur.

Individual genes are transcribed by RNAIndividual genes are transcribed by RNApolymerase II into individual monocistronicpolymerase II into individual monocistronic

mRNA encoding single protein.mRNA encoding single protein.

Proteins that needed to be together willProteins that needed to be together will

not be linked and therefore expressionnot be linked and therefore expression

need to be coordinated.need to be coordinated.


4/23

Genes encoding these proteins are notGenes encoding these proteins are notclosely associated.closely associated.

Often present in different chromosome inOften present in different chromosome inthat particular nucleus.that particular nucleus.

Example the production of antibody byExample the production of antibody bymammalian B cell.mammalian B cell.

Need to synthesize light chain and heavyNeed to synthesize light chain and heavychain to form the antibodychain to form the antibodymolecule/immunoglobulin.molecule/immunoglobulin.


5/23

Genes for antibody:Genes for antibody: light chain in chromosome 2 and 22.light chain in chromosome 2 and 22.

heavy chain in chromosome 14.heavy chain in chromosome 14.

Therefore, greater flexibility neededTherefore, greater flexibility needed

to regulate gene expression into regulate gene expression in

eukaryotes due to this factoreukaryotes due to this factor


6/23

The Britten and DavidsonThe Britten and Davidson

model (1969)model (1969)

This is a model to explain geneThis is a model to explain gene

expression at transcriptional level.expression at transcriptional level.

They proposed that genes regulatedThey proposed that genes regulated

in parallel with one another inin parallel with one another in

response to a particular signalresponse to a particular signal

contain a common regulatorycontain a common regulatory

element.element. Gene will be activated in response toGene will be activated in response to

the signal.the signal.


7/23

Individual genes could contain moreIndividual genes could contain morethan one regulatory element.than one regulatory element.

Some regulatory element would beSome regulatory element would be

shared with other genes.shared with other genes. Specific signal causing geneSpecific signal causing gene

activation where it will stimulateactivation where it will stimulate

specific integrator gene.specific integrator gene.The product of the integrator wouldThe product of the integrator would

activate all the genes containing oneactivate all the genes containing oneparticular sequence element.particular sequence element.


8/23

This mechanism allow the regulationThis mechanism allow the regulationof gene at distinct region.of gene at distinct region.

In modern terms, the integrator geneIn modern terms, the integrator gene

would be considered as transcriptionwould be considered as transcriptionfactor which binds to regulatoryfactor which binds to regulatorysequences and activates expressionsequences and activates expression

of the corresponding genes.of the corresponding genes.

It is clear now that the target DNA-It is clear now that the target DNA-binding sites for transcription factorsbinding sites for transcription factorsare short sequence approximatelyare short sequence approximately

10-30bp in length.10-30bp in length.


9/23

Short sequence elementShort sequence element

The sequences that play role inThe sequences that play role in

regulation of eukaryotic transcriptionregulation of eukaryotic transcription

would be located upstream of thewould be located upstream of the

transcription start site within ortranscription start site within oradjacent to the promoter.adjacent to the promoter.

For example in heat shock protein.For example in heat shock protein.

Gene encoding this protein is calledGene encoding this protein is called

hsp70 with molecular weight ofhsp70 with molecular weight of

70kDa70kDa


10/23

The 70kDa heat-shockThe 70kDa heat-shock

protein geneprotein gene Exposure of a wide variety of cells to highExposure of a wide variety of cells to high

temperature will result in the synthesize oftemperature will result in the synthesize ofheat shock protein , the most abundant isheat shock protein , the most abundant ishsp70.hsp70.

Sequence present in the upstream regionSequence present in the upstream regionofofhsp70hsp70 gene are also found in othergene are also found in othergenes.genes.

The sequences are found shared by :The sequences are found shared by : hsp70hsp70 genegene found in heat-inducible genes.found in heat-inducible genes. Other non heat-inducible genes.Other non heat-inducible genes.


11/23

NameName ConsensusConsensus Other geneOther genecontainingcontaining

sequencesequenceTATA boxTATA box TATAA/TAA/TTATAA/TAA/T In many genesIn many genes

CCAAT boxCCAAT box TGTGGCTNNNAGTGTGGCTNNNAG

CCAACCAA andand -globin,-globin,

herpes simplexherpes simplex

virus thymidinevirus thymidinekinasekinase

Sp1boxSp1box GGGCGGGGGCGG Metallothionein IIMetallothionein II

A. type IIA. type II

procollagenprocollagenCRECRE T/G T/A CGTCAT/G T/A CGTCA Somatostatin,Somatostatin,

fibronectinfibronectin

AP2 boxAP2 box CCCCAGGCCCCCAGGC Collagenase,Collagenase,

class I antigen H-class I antigen H-


12/23

The CCAAT andThe CCAAT and Spl boxesSpl boxes

The basal transcriptional complexThe basal transcriptional complex

containing TBP, RNA polymerase IIcontaining TBP, RNA polymerase II

and other associated factors produceand other associated factors produce

low rate of transcription.low rate of transcription. Rate is enhanced by binding of otherRate is enhanced by binding of other

transcription factors upstream of thetranscription factors upstream of the

TATA box.TATA box.The binding sites of these factors areThe binding sites of these factors are

present in wide variety of gene withpresent in wide variety of gene with

different activity.different activity.


13/23

Example is the Spl box, two copies ofExample is the Spl box, two copies of

which are present in the hsp70 genewhich are present in the hsp70 gene

promoterpromoter

The GC rich DNA sequence binds aThe GC rich DNA sequence binds atranscription factor called Spl which istranscription factor called Spl which is

present in all cell types.present in all cell types.

Another example is CCAAT box which isAnother example is CCAAT box which is

present upstream of the transcription sitepresent upstream of the transcription site

in many genes.in many genes.

These boxes is believed to be involved inThese boxes is believed to be involved in

regulation of transcription by binding toregulation of transcription by binding to


14/23

The heat shock elementThe heat shock element

There are sequence which is sharedThere are sequence which is shared

only by theonly by the hsp70hsp70 gene.gene.

This sequence is found 62 basesThis sequence is found 62 bases

upstream of the start site ofupstream of the start site of

transcription in most heat-inducibletranscription in most heat-inducible

genes.genes.

This heat shock element (HSE) isThis heat shock element (HSE) is

believed to play a critical role in thebelieved to play a critical role in the

transcribing of thetranscribing of the hsp70hsp70 gene.gene.


15/23

To prove this, Pelham (1982)To prove this, Pelham (1982)transferred the HSE on to thetransferred the HSE on to the non-non-heat inducibleheat inducible thymidine kinase (thymidine kinase (tktk))

gene taken from the eukaryotic virus,gene taken from the eukaryotic virus,herpes simplex.herpes simplex.

When hybrid gene introduced intoWhen hybrid gene introduced intocells and temperature raised,cells and temperature raised,increased thymidine kinaseincreased thymidine kinaseproduction was detected.production was detected.

HSE made theHSE made the tktkgene inducible bygene inducible by

elevated temperatureelevated temperature


16/23

hsp 70 tk

tk

tk

Heat inducible

Heat shockConsensus element

Drosophila hsp 70 gene

Non inducible

HSV thymidine kinase gen

Chimaeric gene

Introduced into cellsand raise temperature

Heat inducibletranscription

1:Heat-shock consensus elementtes heat inducibility. Transfer of this sequence

ene (thymidine kinase) which is non induciblers this gene heat inducible


17/23

enhancerenhancer

Regulatory sequences that act at aRegulatory sequences that act at a

distance.distance.

Is an enhancer is linked to a promoter,theIs an enhancer is linked to a promoter,the

activity of the promoter can be increasedactivity of the promoter can be increasedseveral hundred fold.several hundred fold.

The position and orientation of enhancerThe position and orientation of enhancer

results to conclusion of its action:results to conclusion of its action: Enhancer can activate promoter when placedEnhancer can activate promoter when placed

up to several thousand bases from theup to several thousand bases from the

promoter.promoter.

Enhancer can activate promoter regardless ofEnhancer can activate promoter regardless of

its orientation.its orientation.


18/23

Transcription unit

Transcription unit

Transcription unit

Transcription unit

Transcription unit

Transcription unit

Distance enhancer promoter

a) distance

b) orientation

c) position

promoter enhancer

promoter

enhancer

enhancer promoter

promoter

promoter

enhancer

enhancer


19/23

Tissue specific enhancerTissue specific enhancer

A tissue-specific enhancer canA tissue-specific enhancer can

activate the promoter of its own oractivate the promoter of its own or

another gene only in one particularanother gene only in one particular

tissue and not in others.tissue and not in others.Tissue specific enhancers have beenTissue specific enhancers have been

detected in genes expresseddetected in genes expressed

specifically in liver (specifically in liver (-fetoprotein,-fetoprotein,albumin,albumin, -1-antitrypsin) and also the-1-antitrypsin) and also the

endocrine and exocrine cells of theendocrine and exocrine cells of the

pancreas (insulin, elastase, amylase).pancreas (insulin, elastase, amylase).


20/23

In the case of insulin gene, 250 basesIn the case of insulin gene, 250 basesupstream regions of the transcriptionalupstream regions of the transcriptionalstart site being crucial in producing highstart site being crucial in producing highlevel expression in pancreatic endocrinelevel expression in pancreatic endocrinecells.cells.

This position corresponds to the tissueThis position corresponds to the tissuespecific enhancer.specific enhancer.

Therefore this region is important in geneTherefore this region is important in generegulation.regulation. Mutation of the conserved regionMutation of the conserved region

abolishes the production of genes that isabolishes the production of genes that is

usually expressed in that particular tissue.usually expressed in that particular tissue.


21/23

The enhancer and promoter of insulinThe enhancer and promoter of insulingene was linked to gene encoding thegene was linked to gene encoding the

large T antigen of the eukaryotic viruslarge T antigen of the eukaryotic virusSV40.SV40. Production can be measured using specificProduction can be measured using specific

antibody.antibody.

The hybrid was introduced into a fertilizedThe hybrid was introduced into a fertilizedmouse egg and egg was return to themouse egg and egg was return to theoviduct of the mouse.oviduct of the mouse.

Expression of large T antigen analysed inExpression of large T antigen analysed in

all tissue of the transgenic mouse.all tissue of the transgenic mouse. Expression of large T was detected inExpression of large T was detected in

pancreaspancreas and NOT in other tissueand NOT in other tissue Indication of tissue specific activity of theIndication of tissue specific activity of the

insulin gene enhancerinsulin gene enhancer


22/23

Enhancer being silencerEnhancer being silencer

Some sequence act like enhancer butSome sequence act like enhancer butthe other way round.the other way round.

It silent the transcription.It silent the transcription. Example in collagen type II, growthExample in collagen type II, growth

hormone, glutathione transferase.hormone, glutathione transferase. Like enhancers, silencers can act onLike enhancers, silencers can act on

distant promoters when present indistant promoters when present ineither orientation but have aneither orientation but have aninhibitory rather than simulatoryinhibitory rather than simulatoryeffect on the gene expression.effect on the gene expression.


23/23

It acts at chromatin structure byIt acts at chromatin structure byrecruiting factors which direct therecruiting factors which direct thetight packing of adjacent DNA or bytight packing of adjacent DNA or by

binding protein which will then inhibitbinding protein which will then inhibittranscription.transcription.

Or by binding negatively actingOr by binding negatively actingtranscription factors.transcription factors.

Whether acting positively orWhether acting positively ornegatively, enhancers play a criticalnegatively, enhancers play a criticalrole in the regulation of eukaryoticrole in the regulation of eukaryotic

gene expressiongene expression

lect3 transcriptional control

Documents