2. Tryptophan operon.ppt

Nn

1. Arabinose and Tryptophan
Operons
Prof. Rama Swamy Nanna
Department of Biotechnology
Kakatiya University
WARANGAL-TS

2. Theara Operon
Another operon in E. coli that is involved in
sugar
metabolism is the ara (arabinose) operon
It contains
Three structural genes involved in arabinose
metabolism
ThesearedesignatedaraB,araAand araD


Asinglepromoter,P
BAD
ACAPsite,whichbindsthe cataboliteactivator protein

3. The araC gene is adjacent to the ara operon



Ithasits ownpromoter,P
C
Itencodesa regulatoryprotein,AraC
AraCcanbindtothreedifferentoperatorsites
DesignatedaraI,araO
1 and araO
2
TheAraCproteincan actas either
anegativeor positiveregulatorof
transcription
Depending on whetherornot
arabinose is present

4. AraC Negative regulation
AraC protein binds to all three operators
AraCdimer boundto araO
1inhibitstranscriptionof the araCgene
This keepsAraCproteinlevelsfairlylow
AraCmonomers boundto araO
2andaraIrepress the ara operon
They bindtoeach other(vialoopedDNA),and blockRNApolaccessto
P
BAD
araO
1

5. AraC Positive regulation
Arabinose binds to the AraC proteins
Theinteraction betweemtheAraCproteinsat the araO
2andaraIis broken
This breaks theDNAloop
Another,AraCprotein bindsto araI
ThisAraCdimerataraIactivatestranscription
CAP-cAMPactivationoccurs
whenglucoselevelsare low

6. The trp Operon


•The trp operon (pronounced “trip”) is involved in the
biosynthesisof the aminoacid tryptophan
The genestrpE,trpD, trpC, trpB and trpA encode
enzymes involvedin tryptophan biosynthesis
The genestrpR and trpL are involvedin regulation
trpR Encodes the trp repressor protein


Functions in repression
Formsas inactivemonomer, butget activatedwhenattachedto
trp aminoacid andtetramerisethen attachesto oparator.
trpL Encodes a short peptide called the Leader peptide
Functions in attenuation

8. RNApol canbind
tothepromoter
Cannotbindto
theoperatorsite
Organizationof the trp operon and regulation viathetrp
repressor protein

9. TrpOperonONmost of thetime
TrpRgenealsoON:makes inactiverepressorprotein
trpoperon
Promoter Promoter
Genesofoperon
DNA trpR trpE trpD trpC trpB trpA
Operator
Startcodon
StopcodonRegulatory
gene
RNA
polymerase
3
mRNA 5
mRNA
5
E D C B A
Protein Inactive
repressor
Polypeptidesthatmakeup
enzymesfortryptophansynthesis
Tryptophan absent,repressorinactive,operonon

10. Organizationof the trp operon and regulation viathetrp repressorprotein

11. LE18-21b_1
DNA
mRNA
Protein Active
repressor
Tryptophan
(corepressor)
Tryptophan present,repressoractive,operonoff

12. LE18-21b_2
DNA
NoRNAmade
mRNA
Protein Active
repressor
Tryptophan
(corepressor)
Tryptophan present,repressoractive,operonoff

13. Anothermechanism
ofregulation
Organizationof the trp operon and regulation viathetrp repressorprotein

14. Once repression is liftedand transcription begins,the rateof
transcription isfine-tunedbya secondregulatory process,called
transcriptionattenuation,
inwhichtranscriptionisinitiatednormallybutisAbruptly
halted beforetheoperongenesaretranscribed.
Thefrequencywithwhichtranscriptionisattenuatedis
regulatedby the availability oftryptophan andreliesonthe
veryclosecouplingoftranscriptionandtranslationin
Bacteria.

15. Theprocess ofattenuationcomplements repression
regulatoryaction.
Attenuation occurs in bacteria because of the coupling of transcription
andtranslation.
While the TrpR repressor decreases transcription by a factor of 70, attenuation
can further decrease it by a factor of 10, thus allowing accumulated repression
of about 700-fold.
During attenuation, transcription actually begins but it is
terminatedbeforetheentiremRNAis made.



Asegmentof DNA, termed the attenuator, is importantin facilitatingthis
termination
Inthe case of the trp operon,transcriptionterminatesshortlypast thetrpL
region
Thusattenuation inhibitsthe further productionoftryptophan
The segment of trp operon immediately downstream from the operator
siteplays acriticalrolein attenuation
Thefirst genein the trp operonis trpL
It encodes a short peptide termedtheLeaderpeptide

16. •Thisleadertranscriptincludesfourshortsequencesdesignated1-4.
•Sequence1ispartiallycomplementarytosequence2,whichispartially
complementarytosequence3,whichispartiallycomplementaryto
sequence4.
•Thus,threedistinctsecondarystructures(hairpins)canform:1-2,2-3or 3-
4.
•Regulatorysequence1iscrucialforatryptophansensitivemechanism
thatdetermineswhethersequence3pairswithsequence2(allowing
transcriptiontocontinue)or withsequence 4 (attenuating transcription).
•Thehybridizationofstrands1and2toformthe1-2structurepreventsthe
formationofthe2-3structure,whiletheformationof2-3preventsthe
formationof3-4.
Sequence 2is an alternative complementforsequence 3.
If sequences2and3base-pair,theattenuatorstructurecannotformandtranscription
continuesintothe trp biosynthetic genes;the loopformed by thepairingofsequences
2and3 does notobstructtranscription.

17. Region 2 is complementary to regions 1 and 3
Region 3 is complementary to regions 2 and 4
Thereforeseveralstem-loopsstructuresarepossible
The3-4stemloopis
followedby a sequence
ofUracils
Itactsasanintrinsic
(r-independent)terminator
Thesetwocodonsprovidea way
tosenseifthereissufficient
tryptophanavailibilityfor
translation
Sequenceof the trpL mRNAproduced duringattenuation

18. •The3-4structureisatranscriptionterminationsequence,onceitformsRNA
polymerasewilldisassociatefromtheDNAandtranscriptionofthestructural
genesof the operon willnot occur.
•Partoftheleadertranscriptcodesforashortpolypeptideof14aminoacids,
termedthe leaderpeptide.
•Thispeptidecontainstwoadjacenttryptophanresidues,whichisunusual,since
tryptophanisafairlyuncommonaminoacid(aboutoneinahundredresidues
ina typical E. coliproteinistryptophan).
•Iftheribosomeattemptstotranslatethispeptidewhiletryptophanlevelsinthe
cellarelow,it willstall ateitherof the twotrp codons.
•Whileitisstalled,theribosomephysicallyshieldssequence1ofthetranscript,
thus preventingit fromforming the 1-2 secondary structure.
•Sequence2isthenfreetohybridizewithsequence3toformthe2-3structure,
whichthenpreventstheformationofthe3-4terminationhairpin,thusthe2-3
structure is calledanti-termination hairpin.
•RNApolymeraseis freeto continue transcribingthe entire operon.

19. •Iftryptophanlevelsinthecellarehigh,theribosomewilltranslatethe
entireleaderpeptidewithoutinterruptionandwillonlystallduring
translationterminationatthe stopcodon.
•At thispointthe ribosomephysicallyshieldsbothsequences1 and2.
•Sequences3and4arethusfreetoformthe3-4structurewhich
terminatestranscription.
•Theendresultisthattheoperonwillbetranscribedonlywhentryptophan
isunavailablefortheribosome,whilethetrpLtranscriptisconstitutively
expressed.
•Toensurethatthe ribosomebindsand beginstranslationof theleader
transcriptimmediatelyfollowingits synthesis,a pausesite existsin thetrpL
sequence.
•Uponreachingthissite, RNApolymerasepausestranscriptionand
apparentlywaitsfortranslationtobegin.
•Thismechanismallows forsynchronizationoftranscriptionandtranslation,
a key element inattenuation.

20. The formation of the 3-4 stem-loop causes RNA
pol
to terminate transcription at the end of the trpL
gene
Conditions that favor the formation of the 3-4
stem-loop rely on the translation of the trpL mRNA
There are three possible scenarios



1.No translation
2.Low levelsof tryptophan
3.Highlevelsoftryptophan

21. Transcriptionterminates
justpastthe trpLgene
Moststable formof
mRNAoccurs
Thereforeno couplingof
transcriptionand translation

22. Region1 isblocked
3-4stem-loop
doesnot form
RNApol transcribes
restof operon
Insufficientamounts
oftRNA
trp
Sequence 2is an alternative complementforsequence 3.
If sequences2 and 3base-pair,the attenuatorstructurecannotformandtranscription
continuesintothe trp biosynthetic genes;the loopformedby thepairingof sequences
2and 3doesnot obstructtranscription.

24. Sufficientamountsof tRNA
trp
3-4 stem-loopforms
Translationof thetrpLmRNA
progressesuntilstop codon
Transcription
terminates
RNApolymerase pauses
Region2 cannotbasepair
withanyotherregion
Whentryptophanlevelsarehigh,theribosome quicklytranslates sequence1 (open
reading frameencodingleader peptide)and blockssequence 2beforesequence3 is
transcribed.Continuedtranscriptionleads toattenuation at theterminator-like
attenuator structureformed by sequences 3 and4.
attenuatorstructureformed by the pairingof sequences3and 4 (top).Itsstructureand
Functionaresimilartothoseof a transcriptionterminator

26. Induciblevs RepressibleRegulation
The study of many operons revealed a general trend
concerninginducibleversusrepressible regulation
Operons involvedin catabolism(ie. breakdownof a
substance) are typicallyinducible
Thesubstancetobebrokendown(ora relatedcompound)acts
astheinducer
Operons involvedin anabolism(ie. biosynthesisof a
substance) are typicallyrepressible
Theinhibitor orcorepressoris thesmallmoleculethatis the
productofthe operon

27. Repressibleand Inducible Operons:
TwoTypesof NegativeGene Regulation
•A repressibleoperon isone thatis usuallyon;binding
of a repressorshutsofftranscription
•The trp operon isarepressibleoperon
•Aninducibleoperon isone thatisusuallyoff;a
moleculecalled aninducerinactivatestherepressor
andturnson transcription
•The classic exampleofaninducibleoperon isthe lac
operon