Regulation of gene expression in eukaryotes

Mechanism of gene regulation

1. K. Narayanapura, Kothanur (PO), Bengaluru 560077
Tel[phone] Fax. [phone]
e-mail:[email protected], www.kristujayanti.edu.in
Regulation of gene expression in
Eukaryotes
Dr. Manikandan Kathirvel
Assistant Professor,
Department of Life Sciences,
Kristu Jayanti College (Autonomous),
Bengaluru

2. Regulation of Gene Expression in
Eukaryotes
Gene expression is the combined process of :
1.the transcription of a gene into mRNA,
2.the processing of that mRNA, and
3.its translation into protein (for protein-encoding genes).
Levels of regulation of gene expression

3. Purpose of regulation of gene expression :
Regulated expression of genes is required for:
1)Adaptation-Cellsofmulticellularorganisms respondtovarying
conditions.Suchcellsexposed tohormones andgrowthfactors
changesubstantiallyin–shape,growthrateandothercharacteristics
2) Tissue specific differentiation and development
•Thegeneticinformationpresentineachsomaticcellofaorganismis
practicallyidentical.
•Cellsfrommuscleandnervetissueshowstrikinglydifferentmorphologiesand
otherproperties,yettheycontainexactlythesameDNA.
•Thesediversepropertiesaretheresultofdifferencesingene
expression.
•Expression ofthegenetic information isregulated during
developmental stageofanorganismandduringthedifferentiationof
tissuesandbiologicprocessesinthemulticellularorganism.
•Transcriptioncontrolcanresultintissue-specificgeneexpression.

4. Differencesbetweenprokaryoticandeukaryoticgeneexpression:
1)Absence ofoperons:Prokaryote geneexpression typicallyis
regulatedbyanoperon,thecollectionofcontrollingsitesadjacent
topolycistronicprotein-codingsequences.
•Eukaryoticgenesalsoareregulatedinunitsofprotein-coding
sequences andadjacentcontrollingsites,butoperonsarenot
knowntooccur.
2.)Complexity:Eukaryoticgeneregulationismorecomplexbecause
eukaryotespossessanucleus.
3) Differentcelltypes:Differentcelltypesarepresentinmost
eukaryotes.Liverandpancreaticcells,forexample,differdramatically
inthegenesthatarehighlyexpressed.Differentmechanisms are
involvedintheregulationofsuchgenes.
4.)Uncoupledtranscriptionandtranslationprocesses:transcriptionand
translationarenotcoupled).
i.Inprokaryotes,transcriptionandtranslationarecoupledprocesses,theprimarytranscriptis
immediatelytranslated.
ii.Thetranscriptionandtranslationareuncoupledineukaryotes,eliminatingsomepotential
generegulatorymechanisms.
iii.TheprimarytranscriptineukaryotesundergoesmodificationstobecomeamaturefunctionalmRNA.

5. 5)Chromatin structure (in eukaryotes)
-TheDNAineukaryoticcellsisextensivelyfoldedandpackedintothe
protein-DNAcomplexcalledchromatin.
Histonesareanimportantpartofthiscomplexsincetheybothform
thestructuresknownasnucleosomes andalsocontributesignificantly
intogeneregulatorymechanisms.
-Heterochromatin (tanscriptionally inactive)and Euchromatin
(transcriptionallyactive)
Regulation of gene expression in eukaryotes :
Two “categories”of eukaryotic gene regulation exist:
Levels of control of gene expression
Short term control:
Genes are quickly turned on or off in response to the
environment and demands of the cell.
(to meet the daily needs of the organism)
Long term control
(genes for development/differentiation)

6. Mechanism ofregulationofgeneexpression-An
overview
•Geneactivityiscontrolledfirstandforemostatthelevelof
transcription.
•Muchofthiscontrolisachievedthroughtheinterplay
betweenproteinsthatbindtospecificDNAsequencesand
theirDNAbindingsites.
•Thiscanhaveapositiveornegativeeffectontranscription.
•Transcriptioncontrolcanresultintissue-specificgene
expression.
•Inadditiontotranscriptionlevelcontrols,gene
expressioncanalsobemodulated bytranslational
level.
Eukaryotic gene expression is controlled by:
•Cis-Trans acting elements-Transcriptional activation/
repression
•Chromatinmodifications
•Generearrangement,
•Geneamplification,
•Posttranscriptionalmodifications,and
•RNAstabilization
•RegulationbynoncodingRNA
•Posttranslationalmodifications
•ProteinDegradation

7. 1. Cis-Trans acting elements: Transcriptional activation/Repression:
Transcriptional control of gene regulation is controlled by :
a)Promoters
b)Enhancers
c)Silencers
d)Transcriptional factors
e)Activators
f)repressors

8. 1) Cis and Trans acting elements:
Promoters
1. Transcriptional activation:

9. Promoters

10. Promoters
•Occur upstream of the transcription start site.
•Some determine wheretranscription begins (e.g., TATA),
whereas others determine iftranscription begins.
•Promoters are activated by specialized transcription factor (TF)
proteins (specific TFs bind specific promoters).
•Transcription of rRNA –Transcriptional factor-I
•Transcription of mRNA -Transcriptional factor-II
•Transcription of tRNA-Transcriptional factor -III
•One or many promoters (each with specific TF proteins) may
occur for any given gene.
•Promoters may be positively or negatively regulated.

11. EnhancersandRepressors
i.EnhancerelementsareregulatoryDNAsequences,althoughtheyhavenopromoteractivityof
theirownbuttheygreatlyincreasetheactivitiesofmanypromotersineukaryotes.
ii.Enhancersfunctionbyservingasbindingsitesforspecificregulatoryproteinssuchas
transcriptionalfactors.,whenboundbytranscriptionfactors,enhancethetranscriptionofan
associatedgene.
iii.Anenhanceriseffectiveonlyinthespecificcelltypesinwhichappropriateregulatoryproteins
areexpressed.
iv.Enhancerelementscanexerttheirpositiveinfluenceontranscriptionevenwhenseparatedby
thousandsofbasepairsfromapromoter;
v.theyworkwhenorientedineitherdirection;andtheycanworkupstream(5')ordownstream(3')
fromthepromoter.
vi.Enhancersarepromiscuous;theycanstimulateanypromoterinthevicinityandmayactonmore
thanonepromoter.
• Occur upstream or downstream of
the transcription start site or within
the coding sequence
• Regulatory proteins bind specific
enhancer sequences; binding is
determined by the DNA sequence.
• Interactions of regulatory proteins
determine if transcription is activated
or repressed (positively or negatively
regulated).

12. EnhancersandTranscription
Insomeeukaryoticgenes,thereareregions
thathelpincreaseorenhancetranscription.
Theseregions,calledenhancers,arenot
necessarilyclosetothegenestheyenhance.
Theycanbelocatedupstreamofagene,within
thecodingregionofthegene,downstreamofa
gene,ormaybethousandsofnucleotides
away.
Enhancerregionsarebindingsequences,or
sites,fortranscriptionfactors.
WhenaDNA-bendingproteinbindstoan
enhancer,theshapeoftheDNAchanges.This
shapechangeallowstheinteractionbetween
theactivatorsboundtotheenhancersandthe
transcriptionfactorsboundtothepromoter
regionandtheRNApolymerasetooccur.
Therefore,anucleotidesequencethousandsof
nucleotidesawaycanfoldoverandinteract
withaspecificpromoter.
AnenhancerisaDNAsequencethatpromotes
transcription.Eachenhancerismadeupof
shortDNAsequencescalleddistalcontrol
elements.Activatorsboundtothedistal
controlelementsinteractwithmediator
proteinsandtranscriptionfactors.

14. Silencers:
•Theelementsthatdecreaseorrepresstheexpressionofspecificgeneshave
alsobeenidentifiedcalledSilencers.
•SilencersarecontrolregionsofDNAthat,likeenhancers,maybelocated
thousandsofbasepairsawayfromthegenetheycontrol.
•However,whentranscriptionfactorsbindtothem,expressionofthegenethey
controlisrepressed.

15. Trans acting elements

19. MYCgeneencodesamultifunctional,
nuclearphosphoproteinthatcontrolsa
varietyofcellularfunctions,including
cellcycle,cellgrowth,apoptosis,
cellularmetabolismandbiosynthesis,
adhesion, and mitochondrial
biogenesis.

20. Regulation of gene expression in eukaryotes :
Two “categories”of eukaryotic gene regulation exist:
Levels of control of gene expression
Short term control:
Genes are quickly turned on or off in response to the
environment and demands of the cell.
(to meet the daily needs of the organism)
Long term control
(genes for development/differentiation)

21. Short-term -transcriptional control of
galactose-utilizing genes in yeast:
•3 genes (GAL1, GAL7, & GAL 10) code
enzymes that function in the galactose
metabolic pathway.
•GAL1 galactokinase
•GAL7 galactose transferase
•GAL10 galactose epimerase
•Pathway produces d-glucose 6-phosphate,
which enters the glycolytic pathway and is
metabolized by genes that are
continuously transcribed.
•In absence of galactose, GALgenes are not
transcribed.
•GALgenes rapidly induced by galactose
and absence of glucose.
•Analagous to E. colilac operon repression
by glucose.

23. Galactose metabolizing pathway of yeast.

24. Short-term -transcriptional control of galactose-utilizing genes in yeast:
•GALgenes are near each other but do not constitute an operon.
•Additional unlinked gene, GAL4, and GAL80 codes a repressor
protein that binds a promoter element called an upstream activator
sequence (UAS
G).
•UAS
Gis located between GAL1and GAL10.
•Transcription occurs in both directions from UAS
G.
Conditions:
•When galactose is absent, the GAL4product (GAL4p) and another
product (GAL80p) bind the UAS
Gsequence; transcription does not
occur.
•When galactose is added, a galactose metabolite binds GAL80pand
GAL4pamino acids are phosphorylated.
•Galactose acts as an inducer by causing a conformation change in
GAL4p/GAL80p.

25. Activation model of GAL genes in yeast.
Conditions:
• Whengalactoseisabsent,theGAL4
product(GAL4p)andanotherproduct
(GAL80p)bindtheUAS
Gsequence;
transcriptiondoesnotoccur.
Conditions:
• When galactose isadded, agalactose
metabolitebindsGAL80pandGAL4pamino
acidsarephosphorylated.
• Galactoseactsasaninducerbycausinga
conformationchangeinGAL4p/GAL80p.

26. Conditions:
• Whengalactoseisabsent,theGAL4product(GAL4p)andanotherproduct
(GAL80p)bindtheUAS
Gsequence;transcriptiondoesnotoccur.

27. Conditions:
• Whengalactoseisadded,agalactosemetabolitebindsGAL80pand
GAL4paminoacidsarephosphorylated.
• Galactoseactsasaninducerbycausingaconformationchangein
GAL4p/GAL80p.

28. 2)ChromatinRemodeling
•Chromatinstructureprovidesanimportantlevelofcontrolofgene
transcription.
•Thedevelopmentofspecializedorgans,tissues,andcellsandtheirfunctionin
theintactorganismdependuponthedifferentialexpressionofgenes.
•Someofthisdifferentialexpressionisachievedbyhavingdifferentregionsof
chromatinavailablefortranscriptionincellsfromvarioustissues.
Largeregionsofchromatinare
transcriptionallyinactiveinsomecells,
whiletheyareeitheractiveor
potentiallyactiveinotherspecialized
cells.
Forexample,theDNAcontainingthe-
globingeneclusterisin"active"
chromatininthereticulocytesbutin
"inactive"chromatininmusclecells.

29. Formation anddisruptionof
nucleosome structure:
•Thepresenceofnucleosomes
andofcomplexesofhistonesand
DNAprovideabarrieragainstthe
readyassociationoftranscription
factorswithspecificDNAregions.

30. The disruption of nucleosome structure
is therefore an important part of
eukaryotic gene regulation and the
processes involved are as follows:
i) Histone acetylation and deacetylation
Acetylationisknowntooccuronlysine
residuesintheaminoterminaltailsof
histonemolecules.
This modification reduces the positive
charge of these tails and decreases the
binding affinity of histone for the
negatively charged DNA.
Accordingly,theacetylationofhistones
could result in disruption of
nucleosomal structure and allow
readieraccessoftranscriptionfactors
tocognateregulatoryDNAelements.

31. ii) Modification of DNA
MethylationofdeoxycytidineresiduesinDNAmayeffectgrosschanges
inchromatinsoastoprecludeitsactivetranscription.
Example:Acutedemethylationofdeoxycytidineresiduesinaspecificregionof
thetyrosineaminotransferasegene—inresponsetoglucocorticoidhormones—has
beenassociatedwithanincreasedrateoftranscriptionofthegene.

33. iii) DNA binding proteins
•ThebindingofspecifictranscriptionfactorstocertainDNAelementsmayresult
indisruptionofnucleosomalstructure.
•Manyeukaryoticgeneshavemultipleprotein-bindingDNAelements.
•Theserialbindingoftranscriptionfactorstotheseelementsmayeitherdirectly
disruptthestructureofthenucleosomeorpreventitsre-formation.
•Thesereactionsresultinchromatin-levelstructuralchangesthatinthe
endincreaseDNAaccessibilitytootherfactorsandthetranscription
machinery.

34. 3) Gene Amplification
Thegeneproductcanbeincreasedbyincreasingthenumberofgenesavailable
fortranscriptionofspecificmolecules
Duringearlydevelopmentofmetazoans,thereisanabruptincreaseintheneed
forribosomalRNAandmessengerRNAmolecules(hundredsofcopiesof
ribosomalRNAgenesandtRNAgenes)forproteinsthatmakeupsuchorgansas
theeggshell.
Suchrequirementsarefulfilledbyamplificationofthesespecificgenes.
Subsequently,theseamplifiedgenes,presumablygeneratedbyaprocessof
repeatedinitiationsduringDNAsynthesis,providemultiplesitesforgene
transcription.
Geneamplificationhasbeendemonstratedinpatientsreceivingmethotrexatefor
cancer.
Themalignantcellscandevelopdrugresistancebyincreasingthenumberof
genesfordihydrofolatereductase,thetargetofMethotrexate.

35. 4.)mRNAstability
AlthoughmostmRNAs inmammalian cellsareverystable(half-lives
measured inhours),someturnoververyrapidly(half-livesof10–30
minutes).
mRNAstabilityissubjecttoregulation.
Thishasimportant implicationssincethereisusuallyadirect
relationshipbetweenmRNAamountandthetranslationofthatmRNA
intoitscognateprotein.
ChangesinthestabilityofaspecificmRNAcanthereforehavemajor
effectsonbiologicprocesses.
ThestabilityofthemRNAcanbeinfluencedbyhormones andcertain
othereffectors.
TheendsofmRNAmoleculesareinvolvedinmRNAstability.
The5'capstructureineukaryoticmRNA preventsattackby5'
exonucleases, andthepoly(A)tailprohibitstheactionof3'
exonucleases.
PolyAtailing-preventthemRNAfromdegradationandgivesstability
andhelpsthemRNAtotransportfromnucleustocytoplasm
RNAsplicing

36. 5.RegulationofgeneexpressionbyNoncodingRNA
A.Different regions of
mRNA that can be
targeted by an ncRNA
B. Mechanisms of
translation regulation
mediated by ncRNAs
C. RNaseE and RNaseIII
dependent mRNA
degradation mediated by
ncRNAs

37. 6.Alternativesplicing
7.Generearrangement
8.Posttranslationalmodifications
9.ProteinDegradationbyproteasome complex(Ubiquitination)

38. Summary and contrasts :
Prokaryotes control expression by:
Transcription
Eukaryotes control expression by:
Transcription
RNA processing
mRNA transport
mRNA translation
mRNA degradation
Protein degradation
Fig. 18.1

39. Thank you