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6.1.1: Upotreba mutanata za proučavanje lac operona - biologija

6.1.1: Upotreba mutanata za proučavanje lac operona - biologija


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Ciljevi učenja

  • Opišite kako se djelomični diploidi mogu koristiti za ispitivanje genetskih odnosa u haploidnim organizmima.
  • Predvidite da li lac operonski geni će biti eksprimirani kada su prisutne date mutacije.

Pojedinačni mutanti lac operon

The lac operon i njegovi regulatori prvi su put okarakterizirani proučavanjem mutanata E. coli koji su pokazali različite abnormalnosti u metabolizmu laktoze. Neki mutanti su izrazili lac operon geni konstitutivno, što znači da je operon eksprimiran bez obzira na to da li je laktoza bila prisutna u mediju. Takvi mutanti se zovu konstitutivni mutanti.

The položaj operatera (lacO) - Jedan primjer je Oc, u kojoj mutacija u nizu operatora smanjuje ili isključuje represor ( lacI genski proizvod) od prepoznavanja i vezivanja za sekvencu operatora. Dakle, u Oc mutanti, lacZ, lacY, i lacA izraženi su bez obzira na to da li je laktoza prisutna ili ne.

The lacI locus – Jedan tip mutantnog alela lacI (pozvano I-) sprječava ili proizvodnju represorskog polipeptida ili proizvodi polipeptid koji se ne može vezati za sekvencu operatora. Ovo je takođe konstitutivni izraz lac operon jer odsustvo vezivanja represora dozvoljava transkripciju.

Druga vrsta mutanata lacI pozvao Is sprečava represivni polipeptid da veže laktozu, pa će se stoga vezati za operatora i biti neinducibilan. Ovaj mutant konstitutivno potiskuje lac operon bez obzira da li je laktoza prisutna ili ne. The lac operon nije eksprimiran i ovaj mutant se naziva super-represor.

Dva lac operoni u jednoj ćeliji stvaraju parcijalne diploide u E.coli

Više se može saznati o regulaciji lac operon kada su dvije različite kopije prisutne u jednoj ćeliji. To se može postići korištenjem F-faktor da nosi jednu kopiju, dok je druga na genomskoj E. coli hromozom. Ovo rezultira djelomičnim diploidom u E. coli.

F-faktor je ekstra-hromozomska DNK koja može biti ili slobodan plazmid ili integrirana u bakterijski hromozom domaćina. Ovo prebacivanje se postiže IS elementima gdje nejednako ukrštanje može rekombinovati F-faktor i susjedne DNK sekvence (geni) ui izvan hromozoma domaćina. Istraživači su koristili ovaj genetski alat za stvaranje parcijalnih diploida (merozigota) koji im omogućavaju da testiraju regulaciju različitim kombinacijama različitih mutacija u jednoj ćeliji. Na primjer, kopija F-faktora može imati a IS mutacija dok genomska kopija može imati an OC mutacija. Kako bi ova ćelija reagovala na prisustvo/odsustvo laktoze (ili glukoze)? Ovaj parcijalni diploid se može koristiti da se to odredi IS je dominantan za I+, što je zauzvrat dominantno I-. Takođe se može koristiti za prikazivanje OC mutacija djeluje samo u cis- dok je lacI mutacija može djelovati trans- .

Primjer (PageIndex{1})

Razmotrite gore postavljeno pitanje: Ćelija s kopijom lac operon iz F-faktora ima IS mutaciju, dok genomska kopija ima an OC mutacija. Kako bi ova ćelija reagovala na prisustvo/odsustvo laktoze (ili glukoze)?

Rješenje

The IS mutacija kodira oblik proteina koji ne može da veže alolaktozu i stoga nastavlja da prepoznaje operatera, čak i kada je laktoza prisutna. Međutim, genomska kopija operatora OC je mutirana u sekvencu koju više ne prepoznaje lac represor. Iz tog razloga, lacZYA gena u genomu lac operon će biti kontinuirano eksprimiran u ovoj ćeliji, jer lacI represor neće moći da veže ovu sekvencu.


Lac Operan

Pored primarnog operatera, LacO, prikazanog u Slika 1 , dvije 'pseudo-operatorske' sekvence su prisutne unutar lac sekvence operona i doprinose potiskivanju. DNK sekvence pseudo-operatora su vrlo slične, ali ne i identične, LacO i slabije su vezane LacI. Prisustvo dva DNK-vezujuća mjesta u LacI proteinskom tetrameru sugeriralo je mehanizam pomoću kojeg pseudo-operatori mogu pojačati represiju – jedan LacI tetramer može vezati dva odvojena operatora i stvoriti petlju DNK strukturu. Eksperimentalni dokazi za petlju DNK dobiveni su iz raznih laboratorija. Nedavni dokazi pokazuju da se ugao između dva dimera mora otvoriti da bi došlo do formiranja petlje, kao što je ilustrovano u Slika 4 . Ove petljaste strukture su visoko stabilizovane, što predstavlja značajnu represiju lacZYA ekspresija uočena u bakterijskim ćelijama. Zaista, DNK koja sadrži višestruke sekvence operatora i sa karakterističnom gustinom supercoillinga E. coli pokazuje poluživot za kompleks koji prelazi 2 dana. Međutim, čak i ovi kompleksi u petlji brzo reaguju (za manje od 30 s) na prisustvo šećera induktora, omogućavajući brzu adaptaciju na vanjski izvor laktoze koji može biti prolazan.

Slika 4. Petljasta struktura DNK. Plavoplava zakrivljena linija prikazuje lac DNK operona (sa senčenjem za označavanje blizine posmatraču), koji sadrži tri moguća mesta vezanja LacI (od kojih dva, O1 i O2, prikazani su vezani za LacI). Pseudooperatorski niz O2 nalazi se u sklopu lacZ gen, i primarni operator sekvence O1 preklapa sekvencu promotera za lacZYA metabolički geni ( Slika 1 ). Tetramerni LacI je prikazan na dnu slike kao istovremeno u interakciji sa O1 i O2. Ova struktura zamotava DNK i stvara kompleks vrlo visoke stabilnosti. Imajte na umu da se dimeri unutar LacI tetramera razdvajaju i usvajaju veći ugao između njih kada se okreću između O1 i O2 nego u odsustvu petlje (tj. struktura prikazana u Slika 3(a) ). Potreba za fleksibilnošću između dimera da bi došlo do petlje je podržana eksperimentalnim dokazima.


12.1 The lac operon

The lac operon (laktozni operon) je operon potreban za transport i metabolizam laktoze u Escherichia coli i mnoge druge enterične bakterije.

Operaon je funkcionalna jedinica DNK koja sadrži klaster gena pod kontrolom jednog promotora. Geni se zajedno transkribiraju u mRNA lanac i ili se prevode zajedno u citoplazmi, ili se podvrgavaju spajanju kako bi se stvorile monocistronske mRNA koje se transliraju zasebno, tj. nekoliko lanaca mRNA od kojih svaki kodira jedan genski proizvod. Rezultat toga je da se geni sadržani u operonu ili izražavaju zajedno ili uopće ne eksprimiraju. Nekoliko gena mora biti ko-transkribirano da bi se definirao operon.

Iako je glukoza preferirani izvor ugljika za većinu bakterija, lac operon omogućava efikasnu probavu laktoze kada glukoza nije dostupna kroz aktivnost beta-galaktozidaze. Regulacija gena lac operon je bio prvi genetski regulatorni mehanizam koji je jasno shvaćen, tako da je postao najistaknutiji primjer regulacije prokariotskih gena. Iz tog razloga se ovdje detaljno razmatra. Ovaj sistem metabolizma laktoze koristili su François Jacob i Jacques Monod da odrede kako biološka stanica zna koji enzim treba sintetizirati. Njihov rad na lac operon im je donio Nobelovu nagradu za fiziologiju 1965.

Bakterijski operoni su policistronski transkripti koji mogu proizvesti više proteina iz jednog transkripta mRNA. U ovom slučaju, kada je laktoza potrebna kao izvor šećera za bakteriju, tri gena lac operon može biti eksprimiran i njihovi kasniji proteini prevedeni: lacZ, lacY i lacA. Genski proizvod lacZ je β-galaktozidaza koja cijepa laktozu, disaharid, na glukozu i galaktozu. lacY kodira beta-galaktozidnu permeazu, membranski protein koji se ugrađuje u citoplazmatsku membranu kako bi omogućio ćelijski transport laktoze u ćeliju. Konačno, lacA kodira galaktozid acetiltransferazu.

Bilo bi rasipno proizvoditi enzime kada laktoza nije bila dostupna ili kada bi bio dostupan poželjniji izvor energije kao što je glukoza. The lac operon koristi dvodijelni kontrolni mehanizam kako bi osigurao da stanica troši energiju na proizvodnju enzima koje kodira lac operon samo po potrebi. U nedostatku laktoze, lac represor, lacI, zaustavlja proizvodnju enzima koje kodira lac operon. The lac represor je uvijek izražen osim ako se koinduktor ne veže za njega. Drugim riječima, transkribuje se samo u prisustvu koinduktora malih molekula. U prisustvu glukoze, protein aktivator katabolita (CAP), potreban za proizvodnju enzima, ostaje neaktivan, a EIIAGlc isključuje laktoznu permeazu kako bi spriječio transport laktoze u ćeliju. Ovaj dvostruki mehanizam kontrole uzrokuje sekvencijalno korištenje glukoze i laktoze u dvije različite faze rasta, poznate kao diauxie.

Skraćenice od tri slova koriste se za opisivanje fenotipova u bakterijama, uključujući E. coli.

  • lak (sposobnost upotrebe laktoze),
  • Njegova (sposobnost sintetiziranja amino kiseline histidina)
  • Mot (plivačka pokretljivost)
  • SmR (otpornost na antibiotik streptomicin)

U slučaju Lac-a, ćelije divljeg tipa su Lac+ i mogu koristiti laktozu kao ugljik i izvor energije, dok lak-mutantni derivati ​​ne mogu koristiti laktozu. Ista tri slova se obično koriste (mala slova, kurziv) za označavanje gena uključenih u određeni fenotip, pri čemu se svaki različiti gen dodatno razlikuje dodatnim slovom. The lac geni koji kodiraju enzime su lacZ, lacY i lacA. Četvrti lac gen je lacI, koji kodira laktozni represor - "I" označava inducibilnost.

Može se razlikovati između strukturnih gena koji kodiraju enzime i regulatornih gena koji kodiraju proteine ​​koji utiču na ekspresiju gena. Trenutna upotreba proširuje fenotipsku nomenklaturu da se primjenjuje na proteine: tako je LacZ proteinski proizvod lacZ gena, β-galaktozidaze. Različite kratke sekvence koje nisu geni također utiču na ekspresiju gena, uključujući lac promoter, lac p, i lac operater, lac o. Iako nije striktno standardna upotreba, mutacije utiču lac o se nazivaju lac oc, iz istorijskih razloga.

The lac operon se sastoji od 3 strukturna gena i promotora, terminatora, regulatora i operatora. Tri strukturna gena su: lacZ, lacY i lacA.

  • lacZ kodira β-galaktozidazu (LacZ), intracelularni enzim koji cijepa disaharid laktozu na glukozu i galaktozu.
  • lacY kodira Beta-galaktozidnu permeazu (LacY), transmembranski simporter koji pumpa β-galaktozide uključujući laktozu u ćeliju koristeći protonski gradijent u istom smjeru. Permeaza povećava permeabilnost ćelije za β-galaktozide.
  • lacA kodira β-galaktozid transacetilazu (LacA), enzim koji prenosi acetilnu grupu sa acetil-CoA na β-galaktozide.

Čini se da su samo lacZ i lacY neophodni za katabolizam laktoze.

Specifična kontrola lac gena zavise od dostupnosti supstrata laktoze za bakteriju. Bakterija ne proizvodi proteine ​​kada laktoza nije dostupna kao izvor ugljika. The lac geni su organizirani u operon, odnosno orijentirani su u istom smjeru neposredno uz hromozom i ko-transkribirani su u jednu policistronsku mRNA molekulu. Transkripcija svih gena počinje vezivanjem enzima RNA polimeraze (RNAP), proteina koji se vezuje za DNK, koji se vezuje za specifično mesto vezivanja DNK, promotor, odmah uzvodno od gena. Vezivanje RNA polimeraze za promotor je potpomognuto cAMP-vezanim proteinom katabolita aktivator (CAP, također poznat kao protein cAMP receptora). Međutim, lacI gen (regulatorni gen za lac operon) proizvodi protein koji blokira RNAP od vezivanja za promotor operona. Ovaj protein se može ukloniti samo kada se alolaktoza veže za njega i deaktivira ga. Protein koji formira lacI gen poznat je kao the lac represor. Vrsta regulative koju lac operon se naziva negativno inducibilnim, što znači da je gen isključen od strane regulatornog faktora (lac represor) osim ako se doda neki molekul (laktoza). Zbog prisustva lac represor proteina, genetski inženjeri koji zamjene lacZ gen drugim genom morat će uzgajati eksperimentalne bakterije na agaru s dostupnom laktozom. Ako to ne urade, gen koji pokušavaju da eksprimiraju neće biti eksprimiran jer protein represor još uvijek blokira RNAP da se veže za promotor i transkribira gen. Kada se represor ukloni, RNAP zatim nastavlja sa transkripcijom sva tri gena (lacZYA) u mRNA. Svaki od tri gena na mRNA lancu ima svoju Shine-Dalgarno sekvencu, tako da se geni nezavisno prevode. DNK sekvenca E. coli lac operon, lacZYA mRNA i lacI geni su dostupni u GenBank-u (prikaz).

Prvi kontrolni mehanizam je regulatorni odgovor na laktozu, koji koristi intracelularni regulatorni protein nazvan laktozni represor da ometa proizvodnju β-galaktozidaze u odsustvu laktoze. lacI gen koji kodira represor leži u blizini lac operon i uvijek je izražen (konstitutivan). Ako laktoza nedostaje u mediju za rast, represor se vrlo čvrsto vezuje za kratku sekvencu DNK odmah nizvodno od promotora blizu početka lacZ zvanog lac operater. Vezivanje represora za operatera ometa vezivanje RNAP-a za promotor, i stoga se mRNA koja kodira LacZ i LacY stvara samo na vrlo niskim nivoima. Međutim, kada se stanice uzgajaju u prisutnosti laktoze, laktozni metabolit nazvan alolaktoza, napravljen od laktoze produktom lacZ gena, veže se za represor, uzrokujući alosterički pomak. Ovako izmijenjen, represor nije u stanju da se veže za operatera, omogućavajući RNAP-u da transkribira lac gena i na taj način dovodi do viših nivoa kodiranih proteina.

Drugi kontrolni mehanizam je odgovor na glukozu, koji koristi homodimer proteina aktivatora katabolita (CAP) za značajno povećanje proizvodnje β-galaktozidaze u odsustvu glukoze. Ciklični adenozin monofosfat (cAMP) je signalni molekul čija je prevalencija obrnuto proporcionalna onoj glukoze. Veže se za CAP, što zauzvrat omogućava CAP-u da se veže za mjesto vezivanja CAP (sekvenca DNK od 16 bp uzvodno od promotora na donjem dijagramu, oko 60 bp uzvodno od mjesta početka transkripcije), što pomaže RNAP u vezivanju za DNK. U nedostatku glukoze, koncentracija cAMP je visoka i vezivanje CAP-cAMP za DNK značajno povećava proizvodnju β-galaktozidaze, omogućavajući ćeliji da hidrolizuje laktozu i oslobađa galaktozu i glukozu.

Nedavno se pokazalo da isključenje induktora blokira ekspresiju lac operon kada je prisutna glukoza. Glukoza se transportuje u ćeliju putem PEP-ovisnog fosfotransferaznog sistema. Fosfatna grupa fosfoenolpiruvata se prenosi putem kaskade fosforilacije koja se sastoji od općeg PTS (fosfotransferazni sistem) proteina HPr i EIA i glukozno-specifičnih PTS proteina EIIAGlc i EIIBGlc, citoplazmatskog domena prijenosnika glukoze EII. Transport glukoze je praćen njenom fosforilacijom pomoću EIIBGlc, drenirajući fosfatnu grupu iz drugih PTS proteina, uključujući EIIAGlc. Nefosforilirani oblik EIIAGlc se vezuje za lac permeazu i sprečava ga da unese laktozu u ćeliju. Stoga, ako su prisutni i glukoza i laktoza, transport glukoze blokira transport induktora lac operon.

The lac represor je četverodijelni protein, tetramer, sa identičnim podjedinicama (slika 12.2). Svaka podjedinica sadrži motiv spirala-zavoj-heliks (HTH) sposoban da se veže za DNK. Operatorsko mjesto gdje se represor vezuje je DNK sekvenca sa invertiranom simetrijom ponavljanja. Dva polu-mjesta DNK operatora zajedno se vezuju za dvije podjedinice represora. Iako druge dvije podjedinice represora ne rade ništa u ovom modelu, ovo svojstvo nije shvaćeno dugi niz godina.

Na kraju je otkriveno da su u to uključena dva dodatna operatera lac regulacija. Jedan (O3) leži oko -90 bp uzvodno od O1 na kraju lacI gena, a drugi (O2)) je oko +410 bp nizvodno od O1 u ranom dijelu lacZ. Ova dva mjesta nisu pronađena u ranim radovima jer imaju redundantne funkcije i pojedinačne mutacije ne utječu mnogo na potiskivanje. Pojedinačne mutacije na O2) ili O3 imaju samo 2 do 3 puta efekte. Međutim, njihova važnost se pokazuje činjenicom da je dvostruki mutant defektan i u O2) iu O3 dramatično derepresivan (za oko 70 puta).

U trenutnom modelu, lac represor je vezan istovremeno i za glavni operator O1 i za O2) ili O3. Intervenirajuća DNK izlazi iz kompleksa. Redundantna priroda dva manja operatora sugerira da nije važan specifičan kompleks petlje. Jedna ideja je da sistem radi preko povezivanja ako se vezani represor otpusti iz O1 trenutno, vezivanje za manjeg operatera ga drži u blizini, tako da se može brzo ponovo povezati. Ovo bi povećalo afinitet represora za O1.

Slika 12.2: Simulirani strukturni model kompleksa između lac represorski protein (LacI) i 107-bp dug DNK segment. Dvije dimerne LacI funkcionalne podjedinice (zelena +plava i žuta + narandžasta) svaka vezuju sekvencu DNK operatora (gore). Mobilnost grupa glava koje se vezuju za DNK povezane sa stabilnim tijelom LacI-ja obezbjeđuju snagu za petlju DNK (Ville et al.).

Represor je alosterični protein, odnosno može poprimiti bilo koji od dva malo različita oblika, koji su u ravnoteži jedan s drugim. U jednom obliku represor će se vezati za DNK operatera sa visokom specifičnošću, au drugom obliku je izgubio svoju specifičnost. Prema klasičnom modelu indukcije, vezivanje induktora, bilo alolaktoze ili IPTG, za represor utiče na distribuciju represora između dva oblika. Dakle, represor sa vezan induktorom se stabilizuje u konformaciji koja se ne vezuje za DNK. Međutim, ovaj jednostavan model ne može biti cijela priča, jer je represor prilično stabilno vezan za DNK, ali se brzo oslobađa dodavanjem induktora. Stoga se čini jasnim da se induktor također može vezati za represor kada je represor već vezan za DNK. Još uvijek nije sasvim poznato koji je tačan mehanizam vezivanja.

Nespecifično vezivanje represora za DNK igra ključnu ulogu u potiskivanju i indukciji Lac-operona. Specifično mjesto vezivanja za Lac-represorski protein je operater. Nespecifična interakcija je posredovana uglavnom interakcijama naboj-naboj, dok je vezivanje za operatera pojačano hidrofobnim interakcijama. Osim toga, postoji obilje nespecifičnih DNK sekvenci za koje se represor može vezati. U suštini, svaka sekvenca koja nije operator, smatra se nespecifičnom. Istraživanja su pokazala da bez prisustva nespecifičnog vezivanja, indukcija (ili nerepresija) Lac-operona ne bi mogla da se dogodi čak ni sa zasićenim nivoima induktora. Pokazalo se da je, bez nespecifičnog vezivanja, bazalni nivo indukcije deset hiljada puta manji od uobičajenog. To je zato što nespecifična DNK djeluje kao neka vrsta "slivnika" za proteine ​​represora, odvlačeći ih od operatera. Nespecifične sekvence smanjuju količinu raspoloživog represora u ćeliji. Ovo zauzvrat smanjuje količinu induktora koja je potrebna za depresiju sistema.

Opisani su brojni derivati ​​ili analozi laktoze koji su korisni za rad sa lac operon. Ova jedinjenja su uglavnom supstituisani galaktozidi, gde je glukozni deo laktoze zamenjen drugom hemijskom grupom. Izopropil-β-D-tiogalaktopiranozid (IPTG) se često koristi kao induktor lac operon za fiziološki rad. IPTG se vezuje za represor i inaktivira ga, ali nije supstrat za β-galaktozidazu. Jedna od prednosti IPTG-a za in vivo studije je to što se ne može metabolizirati E. coli njegova koncentracija ostaje konstantna i stopa ekspresije lac p/o-kontrolisani geni, nije varijabla u eksperimentu. Unos IPTG zavisi od delovanja laktozne permeaze u P. fluorescens, ali ne i u E. coli.

Eksperimentalni mikroorganizam koji su koristili François Jacob i Jacques Monod bila je uobičajena laboratorijska bakterija, E. coli, ali mnogi od osnovnih regulatornih koncepta koje su otkrili Jacob i Monod su fundamentalni za ćelijsku regulaciju u svim organizmima. Ključna ideja je da se proteini ne sintetiziraju kada nisu potrebni – E. coli čuva ćelijske resurse i energiju tako što ne proizvodi tri Lac proteina kada nema potrebe za metabolizmom laktoze, kao kada su dostupni drugi šećeri poput glukoze. Sljedeći odjeljak govori o tome kako E. coli kontroliše određene gene kao odgovor na metaboličke potrebe.

Tokom Drugog svetskog rata, Monod je testirao efekte kombinacija šećera kao izvora hranljivih materija E. coli i B. subtilis. Monod je pratio slične studije koje su proveli drugi naučnici sa bakterijama i kvascem. Otkrio je da bakterije uzgajane s dva različita šećera često pokazuju dvije faze rasta. Na primjer, ako su obezbijeđene glukoza i laktoza, glukoza se prvo metabolizira (faza rasta I, vidi sliku 2), a zatim laktoza (faza rasta II). Laktoza se nije metabolizirala tokom prvog dijela dijauksičke krivulje rasta jer β-galaktozidaza nije stvorena kada su i glukoza i laktoza bile prisutne u mediju. Monod je ovaj fenomen nazvao diauxie.

Monod je zatim usmjerio svoju pažnju na indukciju stvaranja β-galaktozidaze do koje je došlo kada je laktoza bila jedini šećer u mediju kulture.

Konceptualni proboj Jacoba i Monoda bio je da prepoznaju razliku između regulatornih supstanci i mjesta na kojima djeluju da mijenjaju ekspresiju gena. Bivši vojnik, Jacob je koristio analogiju bombardera koji bi oslobodio svoj smrtonosni teret po prijemu specijalnog radio prijenosa ili signala. Za radni sistem potrebni su i zemaljski predajnik i prijemnik u avionu. Sada, pretpostavimo da je uobičajeni predajnik pokvaren. Ovaj sistem se može učiniti da radi uvođenjem drugog, funkcionalnog predajnika. Nasuprot tome, rekao je, razmislite o bombarderu sa neispravnim prijemnikom. Ponašanje ovog bombardera ne može se promeniti uvođenjem drugog, funkcionalnog aviona.

Za analizu regulatornih mutanata lac operon, Jacob je razvio sistem pomoću kojeg je druga kopija lac geni (lacI sa svojim promotorom, i lacZYA sa promotorom i operatorom) mogu se uvesti u jednu ćeliju. Kultura takvih bakterija, koje su diploidne za lac gena, ali inače normalnih, zatim se testira na regulatorni fenotip. Konkretno, utvrđuje se da li se LacZ i LacY stvaraju čak iu odsustvu IPTG-a (zbog nefunkcionalnog represora laktoze koji proizvodi mutantni gen). Ovaj eksperiment, u kojem se geni ili klasteri gena testiraju u paru, naziva se komplementacijski test.

Ovaj test je ilustrovan na slici 12.4 (lacA je izostavljen radi jednostavnosti). Prvo, prikazana su određena haploidna stanja (tj. ćelija nosi samo jednu kopiju lac geni). Panel (a) prikazuje represiju, (b) prikazuje indukciju IPTG-om, i (c) i (d) pokazuju efekat mutacije na lacI gen ili na operatera, respektivno. Na panelu (e) prikazan je test komplementacije za represor. Ako jedan primjerak lac geni nose mutaciju u lacI, ali druga kopija je divljeg tipa za lacI, rezultirajući fenotip je normalan—ali lacZ se eksprimira kada je izložen induktoru IPTG. Za mutacije koje utječu na represor se kaže da su recesivne na divlji tip (i taj divlji tip je dominantan), a to se objašnjava činjenicom da je represor mali protein koji može difundirati u ćeliji. Kopija lac operon pored defektnog lacI gena je efikasno isključen proteinom proizvedenim iz druge kopije lacI.

Ako se isti eksperiment izvede upotrebom mutacije operatora, dobije se drugačiji rezultat (panel (f)). Fenotip ćelije koja nosi jedno mutantno i jedno operatorsko mjesto divljeg tipa je da se LacZ i LacY proizvode čak i u odsustvu induktora IPTG jer oštećeno operatorsko mjesto ne dozvoljava vezivanje represora da inhibira transkripciju strukturnih gena. Mutacija operatora je dominantna. Kada je mjesto operatera gdje se represor mora vezati oštećeno mutacijom, prisustvo drugog funkcionalnog mjesta u istoj ćeliji ne čini nikakvu razliku u ekspresiji gena koje kontrolira mutantno mjesto.

Sofisticiranija verzija ovog eksperimenta koristi označene operone za razlikovanje između dvije kopije lac geni i pokazuju da je(i) neregulisani strukturni geni(ovi) onaj(ovi) pored mutantnog operatora (panel (g). Na primjer, pretpostavimo da je jedna kopija označena mutacijom koja inaktivira lacZ tako da može samo proizvode LacY protein, dok druga kopija nosi mutaciju koja utječe na lacY i može proizvesti samo LacZ. U ovoj verziji, samo kopija proteina lac operon koji je u blizini mutantnog operatora je izražen bez IPTG. Kažemo da je mutacija operatora cis-dominantna, dominantna je na divlji tip, ali utiče samo na kopiju operona koja je neposredno uz nju.

Ovo objašnjenje je pogrešno u važnom smislu, jer polazi od opisa eksperimenta, a zatim objašnjava rezultate u terminima modela. Ali u stvari, često je istina da je model na prvom mjestu, a eksperiment je napravljen posebno za testiranje modela. Jacob i Monod su prvo zamislili da mora postojati mjesto u DNK sa svojstvima operatora, a zatim su dizajnirali svoje komplementarne testove da to pokažu.

Dominacija operatorskih mutanata također sugerira proceduru za njihov poseban odabir. Ako su regulatorni mutanti odabrani iz kulture divljeg tipa koristeći fenil-Gal, kao što je gore opisano, mutacije operatera su rijetke u usporedbi s represorskim mutantima jer je ciljna veličina tako mala. Ali ako umjesto toga krenemo od soja koji nosi dvije kopije cjeline lac regiji (koja je diploidna za lac), mutacije represora (koje se još uvijek javljaju) nisu pronađene jer komplementacija drugim, divljim tipom lacI gena daje fenotip divljeg tipa. Nasuprot tome, mutacija jedne kopije operatora daje mutantni fenotip jer je dominantan u odnosu na drugu kopiju divljeg tipa.

Objašnjenje diauxie ovisi o karakterizaciji dodatnih mutacija koje utječu na lac gene koji nisu objašnjeni klasičnim modelom. Dva druga gena, cya i crp, naknadno su identifikovana koji su mapirani daleko od lac, i koji, kada su mutirani, dovode do smanjenog nivoa ekspresije u prisustvu IPTG-a, pa čak i kod sojeva bakterije kojima nedostaje represor ili operator. Otkriće cAMP u E. coli doveo je do demonstracije da se mutanti koji defektiraju cya gen, ali ne i crp gen, mogu vratiti u punu aktivnost dodavanjem cAMP u mediju.

Cia gen kodira adenilat ciklazu, koja proizvodi cAMP. Kod cya mutanta, odsustvo cAMP čini ekspresiju lacZYA gena više od deset puta nižom od normalne. Dodavanje cAMP ispravlja nisku ekspresiju Lac karakterističnu za cya mutante. Drugi gen, crp, kodira protein koji se naziva protein aktivator katabolita (CAP) ili protein cAMP receptora (CRP).

Međutim, enzimi metabolizma laktoze nastaju u malim količinama u prisutnosti i glukoze i laktoze (ponekad se naziva nepropusna ekspresija) zbog činjenice da se LacI represor brzo povezuje/odvaja od DNK umjesto da se čvrsto vezuje za nju, što može omogućiti vrijeme da RNAP veže i transkribira mRNA lacZYA. Nepropusna ekspresija je neophodna kako bi se omogućio metabolizam neke laktoze nakon što se izvor glukoze potroši, ali prije lac ekspresija je potpuno aktivirana.

  • Kada je laktoza odsutna, postoji vrlo mala proizvodnja Lac enzima (operater ima vezan za Lac represor).
  • Kada je laktoza prisutna, ali je također prisutan preferirani izvor ugljika (kao što je glukoza), tada se proizvodi mala količina enzima (Lac represor nije vezan za operatera).
  • Kada je glukoza odsutna, CAP-cAMP se vezuje za specifično mjesto DNK uzvodno od promotora i ostvaruje direktnu interakciju protein-protein sa RNAP-om što olakšava vezivanje RNAP-a za promotor.

Kašnjenje između faza rasta odražava vrijeme potrebno za proizvodnju dovoljnih količina enzima koji metaboliziraju laktozu. Prvo, CAP regulatorni protein se mora sastaviti na lac promotor, što rezultira povećanjem proizvodnje lac mRNA. Više dostupnih primjeraka lac mRNA rezultira proizvodnjom (vidi prijevod) znatno više kopija LacZ (β-galaktozidaze, za metabolizam laktoze) i LacY (laktozna permeaza za transport laktoze u ćeliju). Nakon odgode potrebnog za povećanje razine enzima koji metaboliziraju laktozu, bakterije ulaze u novu brzu fazu rasta stanica.

Dve zagonetke potiskivanja katabolita odnose se na to kako su nivoi cAMP-a povezani sa prisustvom glukoze, i drugo, zašto bi ćelije uopšte trebale da smetaju. Nakon što se laktoza cijepa, ona zapravo stvara glukozu i galaktozu (lako se pretvara u glukozu). U metaboličkom smislu, laktoza je jednako dobar ugljik i izvor energije kao i glukoza. Nivo cAMP nije povezan sa intracelularnom koncentracijom glukoze, već sa brzinom transporta glukoze, koja utiče na aktivnost adenilat ciklaze. (Pored toga, transport glukoze također dovodi do direktne inhibicije laktozne permeaze.) Zašto E. coli radi na ovaj način, može se samo nagađati. Sve enterične bakterije fermentiraju glukozu, što sugerira da se s njom često susreću. Moguće je da mala razlika u efikasnosti transporta ili metabolizma glukoze protiv laktoze čini korisnim za ćelije da regulišu lac operon na ovaj način.

The lac gen i njegovi derivati ​​se mogu koristiti kao reporterski gen u brojnim tehnikama selekcije zasnovanim na bakterijama, kao što su dvije hibridne analize, u kojima se mora utvrditi uspješno vezivanje transkripcijskog aktivatora za specifičnu promotorsku sekvencu. U LB pločama koje sadrže X-gal, promjena boje od bijelih kolonija do nijanse plave odgovara oko 20-100 jedinica β-galaktozidaze, dok tetrazolium laktoza i MacConkey laktozni mediji imaju raspon od 100-1000 jedinica, najosjetljiviji su u visoki i donji dio ovog raspona respektivno. Budući da se MacConkey laktozni i tetrazolij laktozni medij oslanjaju na produkte razgradnje laktoze, zahtijevaju prisustvo i lacZ i lacY gena. Mnogi lac fuzijske tehnike koje uključuju samo lacZ gen su stoga pogodne za X-gal ploče ili ONPG tečne bujone. ONPG (orto-nitrofenil-β-galaktozid) je kolorimetrijski i spektrofotometrijski supstrat za detekciju aktivnosti β-galaktozidaze. Ovo jedinjenje je normalno bezbojno. Međutim, ako je prisutna β-galaktozidaza, ona hidrolizira ONPG molekulu u galaktozu i orto-nitrofenol. Potonje jedinjenje ima žutu boju koja se može koristiti za provjeru aktivnosti enzima kolorimetrijskim testom (na talasnoj dužini od 420 nm). β-galaktozidaza je potrebna za iskorištavanje laktoze, tako da se intenzitet proizvedene boje može koristiti kao mjera enzimske brzine. Iako ONPG oponaša laktozu i hidrolizira ga β-galaktozidaza, on nije u stanju djelovati kao induktor za lac operon. Bez drugog analoga laktoze koji može djelovati kao induktor, kao što je izopropil β-D-1-tiogalaktopiranozid (IPTG), β-galaktozidaza neće biti transkribirana i ONPG neće biti hidrolizovan.


Priroda konstitutivnih mutacija operatora laktoze ☆,☆☆

Analiza velikog broja O c mutanti lac operona pružili su dokaze koji ukazuju na to da: (a) prevlast O c mutacije su baznog supstitucijskog tipa i sve daju samo djelomičnu konstitutivnost za sintezu Lac enzima (b) gotovo sve u jednom koraku O c mutacije spadaju u jednu ili drugu od malog broja klasa u odnosu na konstitutivne nivoe (c) mnoge O c mutacije također imaju prateće promotorske efekte, tj utiču na maksimalan nivo sinteze Lac enzima.

Ovaj rad je podržan od strane Nacionalnog instituta za zdravlje s brojevima grantova GM13383 i GM16308, dok je jedan od nas (JRS) bio dobitnik nagrade za razvoj karijere, GM-28123, a drugi (TS) je dobio Nacionalnu zdravstvenu postdoktorsku stipendiju HD 42801 .

Ovo je doprinos br. 415 sa Instituta Eleanor Roosevelt za istraživanje raka i Odeljenja za biofiziku Medicinskog centra Univerziteta Kolorado, Denver, Kolorado.


Pitanja kritičkog razmišljanja

Sve ćelije jednog organizma dijele genom. Međutim, tokom razvoja, neke ćelije se razvijaju u ćelije kože, dok se druge razvijaju u mišićne ćelije. Kako iste genetske upute mogu rezultirati dvije različite vrste stanica u istom organizmu? Detaljno obrazložite svoj odgovor.

Različiti genetski programi se uključuju ili isključuju kada se ćelije diferenciraju u različite tipove ćelija (npr. ćelije kože, mišićne ćelije, itd.) Kao rezultat, ćelije izražavaju gene potrebne za tkivo u kojem se nalaze.

  1. Kada su laktoza i glukoza prisutni u mediju, indukuje se transkripcija lac operona.
  2. Kada je laktoza prisutna, ali je glukoza odsutna, lac operon je potisnut.
  3. Lactose acts as an inducer of the lac operon when glucose is absent.
  4. Lactose acts as an inducer of the lac operon when glucose is present.
  1. Mutation in structural genes will stop transcription.
  2. Mutated lacY will prevent CAP from binding.
  3. Mutated lacA will metabolize lactose or maltodextrin.
  4. Transcription will continue but lactose will not be metabolized properly.

In some diseases, alteration to epigenetic modifications turns off genes that are normally expressed. Hipotetički, kako biste mogli preokrenuti ovaj proces da biste ponovo uključili ove gene?

  1. In new seedlings, histone acetylations are present upon cold exposure, methylation occurs.
  2. In new seedlings, histone deacetylations are present upon cold exposure, methylation occurs.
  3. In new seedlings, histone methylations are present upon cold exposure, acetylation occurs.
  4. In new seedlings, histone methylations are present upon cold exposure, deacetylation occurs.
  1. Mutated promoters decrease the rate of transcription by altering the binding site for the transcription factor.
  2. Mutated promoters increase the rate of transcription by altering the binding site for the transcription factor.
  3. Mutated promoters alter the binding site for transcription factors to increase or decrease the rate of transcription.
  4. Mutated promoters alter the binding site for transcription factors and thereby cease transcription of the adjacent gene.
  1. The transcription rate would increase, altering cell function.
  2. The transcription rate would decrease, inhibiting cell functions.
  3. The transcription rate decreases due to clogging of the transcription factors.
  4. The transcription rate increases due to clogging of the transcription factors.

The wnt transcription pathway is responsible for key changes during animal development. Based on the transcription pathway shown below. In this diagram, arrows indicate the transformation of one substance into another. Square lines, or the lines with no arrowheads, indicate inhibition of the product below the line. Based on this, how would increased wnt gene expression affect the expression of Bar-1?

  1. RBPs can bind first to the RNA, thus preventing the binding of miRNA, which degrades RNA.
  2. RBPs bind the miRNA, thereby protecting the mRNA from degradation.
  3. RBPs methylate miRNA to inhibit its function and thus stop mRNA degradation.
  4. RBPs direct miRNA degradation with the help of a DICER protein complex.
  1. UV rays can alter methylation and acetylation of proteins.
  2. RNA binding proteins are modified through phosphorylation.
  3. External stimuli can cause deacetylation and demethylation of the transcript.
  4. UV rays can cause dimerization of the RNA binding proteins.
  1. Phosphorylation of proteins can alter translation, RNA shuttling, RNA stability or post transcriptional modification.
  2. Phosphorylation of proteins can alter DNA replication, cell division, pathogen recognition and RNA stability.
  3. Phosphorylated proteins affect only translation and can cause cancer by altering the p53 function.
  4. Phosphorylated proteins affect only RNA shuttling, RNA stability, and post-translational modifications.
  1. UV rays could cause methylation and deacetylation of the genes that could alter the accessibility and transcription of DNA.
  2. The UV rays could cause phosphorylation and acetylation of the DNA and histones which could alter the transcriptional capabilities of the DNA.
  3. UV rays could cause methylation and phosphorylation of the DNA bases which could become dimerized rendering no accessibility of DNA.
  4. The UV rays can cause methylation and acetylation of histones making the DNA more tightly packed and leading to inaccessibility.
  1. These drugs maintain the demethylated and the acetylated forms of the DNA to keep transcription of necessary genes “on”.
  2. The demethylated and the acetylated forms of the DNA are reversed when the silenced gene is expressed.
  3. The drug methylates and acetylates the silenced genes to turn them back “on”.
  4. Drugs maintain DNA methylation and acetylation to silence unimportant genes in cancer cells.
  1. Understanding gene expression patterns in cancer cells will identify the faulty genes, which is helpful in providing the relevant drug treatment.
  2. Understanding gene expression will help differentiate cancerous cells from malignant cells.
  3. Gene profiling would identify the target genes of the cancer-causing bacteria.
  4. Breast cancer patients who do not express EGFR can respond to anti-EGFR therapy.
  1. Personalized medicines would vary based on the type of mutations and the gene’s expression pattern.
  2. The medicines are given based on the type of tumor found in the body of an individual.
  3. The personalized medicines are provided based only on the symptoms of the patient.
  4. The medicines tend to vary depending on the severity and the stage of the cancer.

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    • Autori: Julianne Zedalis, John Eggebrecht
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    BIOL 3301 EXAM 3 Section 07869 GENETICS Wednesday April 2 2003 1 00 2 30 pm NOTE MAKE SURE YOU READ THE QUESTIONS CAREFULLY Name SSN True false statements write True or False in front of the statement 2 points each 1 The experiments by Griffith on Streptococcus pneumoniae were the first demonstration of bacterial transformation T 2 DNA polymerase III is the main activity during DNA replication T 3 Helicase is required for synthesis of the RNA primer during DNA replication F 4 Topoisomerase is a reverse transcriptase that maintains chromosome ends F 5 DNA ligase catalyzes the formation of hydrogen bonds F 6 The RNA polymerase core enzyme is sufficient for correct initiation of transcription F 7 Protein synthesis is terminated by a rho dependent mechanism F 8 IF2 stimulates binding of the 30S ribosome subunit to mRNA F 9 A homeodomain is a cis regulatory sequence F 10 Enhancers can only operate upstream of promoters they are enhancing F Multiple choice circle the correct answer 2 points each 1 A sample of normal double stranded DNA was found to have a thymine content of 27 What is the expected proportion of guanine a 9 b 23 c 32 d 36 e 73 2 The figure shown below depicts the structure of a dATP b dCTP c ddATP d UTP e ddTTP 3 Hershey and Chase proved that DNA is the transforming principle They observed that the isotope accumulated in the bacteria was a 31P b 35S c 32P d 15N e none of the above 4 In a DNA double helix nucleotides on complementary strands are held together by a covalent bonds b salt bridges c hydrogen bonds d phosphodiester bonds e none of the above 5 Meselsohn and Stahl demonstrated that DNA replication is a conservative b dispersive c funky d semi conservative e none of the above 6 rII mutants are grown on the restrictive host E coli K plate 1 and on the permissive host E coli B plate 2 The plaques appear as follows a Plate 1 no plaques Plate 2 large plaques b Plate 1 large plaques Plate 2 large plaques c Plate 1 small plaques Plate 2 small plaques d Plate 1 no plaques Plate 2 small plaques e Plate 1 no plaques Plate 2 no plaques 7 Mutations that do not complement are a allelic b non allelic c intergenic d null mutants e missense mutations 8 A tRNA with the anticodon 3 UGG 5 would carry a phenylalanine b tyrosine c tryptophane d serine e threonine 9 The E coli RNA polymerase holoenzyme consists of the following subunits a 2 1 1 1 b 2 2 1 c 2 1 1 d 1 2 1 1 e 2 2 1 10 The following diagram shows a fragment of transcribed DNA and the upper strand is the template strand 5 ATTGCC 3 3 TAACGG 5 The transcribed RNA can be represented by a 5 AUUGCC 3 b 5 TAACGG 3 c 5 AUUGCC 3 d 5 UAACGG 3 e 5 GGCAAU 3 11 EF Tu is required for a loading aminoacyl tRNA into the A site of the ribosome b regenerating EF Ts c translocation of the peptidylchain d translocation of the ribosome e releasing empty tRNAs from the ribosome 12 Stop codons are not recognized by tRNAs but by a ribosomes b the Shine Dalgarno sequence c release factors d EF Tu e IF3 13 A partial diploid of genotype I P O Z Y I P O Z Y will show a inducible production of beta galactosidase b inducible production of beta galactosidase and permease c constitutive production of beta galactosidase and permease d inducible production of permease e no beta galactosidase or acetylase production at all 14 Which of the following statements is true for eukaryotic RNA polymerases a RNA polymerase I synthesizes mRNA b RNA polymerase II synthesizes snRNA and rRNA c RNA polymerase I synthesizes rRNA d RNA polymerase III synthesizes mRNA e none of the above 15 Transcription termination in eukaryotes depends on a a hairpin structure b Rho termination factor c recognition of a AAUAAA sequence and endonuclease cleavage d polyadenylation e guanyltransferase Fill in 2 points each 1 Two of the bases Adenine and Guanine are similar in structure and are called purines The other two bases Thymine and Cytosine are called pyrimidines 2 B DNA is the most common form of DNA The spacing between basepairs is 3 4 A 3 Yanofsky studied the trp synthetase gene and demonstrated the colinearity of gene and protein 4 E coli DNA replication begins from a fixed origin but then proceeds bidirectionally ending at a site called the terminus 5 Translation initiation depends on recognition of the Shine Dalgarno sequence by the 3 end of 16S rRNA Homework 1 A We are studying some of the Neurospora mutants described by Beadle and Tatum These mutants numbered 1 to 7 are all involved in the same biosynthetic pathway The compounds generated in the different biosynthetic steps are A B C D F G H I Reconstruct the biosynthetic pathway starting on the left with the compound that comes first in the biosynthetic pathway 7 points Compound tested growth no growth A B C D F G H I 1 2 3 4 5 6 7 H G I A D F B C 4 7 3 2 5 6 1 B What are auxotrophic mutants 3 points Mutants that require addition of a supplement to grow on minimal medium 2 You are studying a gene in E coli that specifies a protein A part of its sequence is Phe Tyr Ser Trp Glu Ala Met ThrYou recover a series of mutants for this gene that show no enzymatic activity By isolating the mutant enzyme products you find the following sequences Mutant 1 Phe Asn Ser Trp Glu Ala Met Thr Mutant 2 Phe Tyr Ser Trp Mutant 3 Phe His Cys Leu Pro Ala Val Thr Mutant 4 Phe Tyr Met Leu Gly Ala Met Thr What is the DNA sequence that specifies this part of the protein What is the molecular nature for each mutation 10 points Wildtype TTC TAT TCN TGG GAA GCN ATG ACN C C AGT G C Mutant 1 TTC AAT TCN TGG GAA GCN ATG ACN C C AGT G C Point mutation leads to amino acid substitution Mutant 2 TTC AAT TCN TGG TAA C C AGT C Point mutation leads to stop codon Mutant 3 TTC CAT TGT CTN CCN GCN GTN ACN C C C TTA G Inversion Mutant 4 TTC TAT ATG CTN GGN GCN ATG ACN C C TTA G Insertion single underlined and deletion double underlined Open questions 20 points NOTE If you would run out of space you can also use the back of this page to continue your answer Describe and explain the lac operon and its regulation including catabolite repression LAC OPERON STRUCTURE The lac operon consists of POZYA P O are cis regulatory sequences P promoter O operator ZYA are structural genes lacZ encodes beta galactosidase lacY encodes permease lacA encodes transacetylase Beta galactosidase converts lactose into glucose and …


    Important topics of solutions for NCERT class 12 biology chapter 6 molecular basis of inheritance:

    6.1.1 Structure of Polynucleotide Chain

    6.1.2 Packaging of DNA Helix

    6.2 The Search for Genetic Material

    6.2.1 The Genetic Material is DNA

    6.2.2 Properties of Genetic Material (DNA versus RNA)

    6.4.1 The Experimental Proof

    6.4.2 The Machinery and the Enzymes

    6.5.2 Transcription Unit and the Gene

    6.5.3 Types of RNA and the process of Transcription

    6.6.1 Mutations and Genetic Code

    6.6.2 tRNA– the Adapter Molecule

    6.8 Regulation of Gene Expression

    6.9.1 Salient Features of Human Genome

    6.9.2 Applications and Future Challenges

    In CBSE NCERT solutions for class 12 biology chapter 6 molecular basis of inheritance, you will also get solutions to the questions based on the human genome project as it was a megaproject that aimed to sequence every base in the human genome. This project has yielded much new information among us. Many new areas and avenues have opened up as a consequence of the project. In NCERT solutions for class 12 biology chapter 6 molecular basis of inheritance, you will get an explanation of DNA Fingerprinting. It is a technique to find out variations in individuals of a population at the DNA level. It works on the principle of polymorphism in DNA sequences.

    In solutions for NCERT class 12 biology chapter 6 molecular basis of inheritance, you will get solutions to the questions based on two important nucleic acid which is also called as genetic materials for living organisms and these are:

    DNA and RNA are the two types of nucleic acids found in living systems whereas DNA is double-stranded and RNA is single-stranded, DNA acts as the genetic material in most of the organisms and RNA acts as genetic material in some viruses, mostly functions as a messenger. In NCERT solutions for class 12 biology chapter 6 molecular basis of inheritance, you will get solutions to the questions on Central dogma which consists of three important things that are:

    After going through solutions for NCERT class 12 biology chapter 6 molecular basis of inheritance you will be able to answer all the questions which are given at the end of this chapter.


    MATERIJALI I METODE

    Preparation of proteins and DNA

    Lac repressor (both wild-type and mutants) was overexpressed in BLIM cells (22) and purified according to Chen and Matthews (23). The DNA construct used in TPM experiments was synthesized by PCR similarly to the method described by Finzi and Gelles (20). Briefly, the pRW490 plasmid (19), containing two primary operators (with sequence 5′-TGTTGTGTGGAATTGTGAGCGGATAACAAT-TTCACACAGG-3′) spaced 305 bp apart, was used as template in a PCR (201223, Qiagen, Germany) employing the following primers (MWG-Biotech AG, Germany): 5′-Biotin-AGATCCAGTTCGATGT-3′ 5′-Digoxigenin-ATAGTGGCTCCAAGTAGC-3′.

    The PCR product (purified using 28104, Qiagen, Germany) is a 1302 bp molecule labeled with biotin at one end and with digoxigenin at the other end (see Figure 1 ).

    A flow chamber (with a volume of � μl) was constructed between a microscope slide and a coverslip kept at a distance of � μm by double-sided tape. Strips of silicon grease were deposited on the inner side of the tape to provide lateral sealing and avoid contact of the solution with the tape itself. The microscope slides were previously cleaned in an ultrasonic bath in ethanol for 5 min. The coverslips were cleaned further by a 10 min treatment with reactive ion plasma in a radio-frequency plasma cleaner (PDC-002, Harrick Inc., NY).

    Buffers and sample preparation for TPM

    Unless otherwise specified, all chemicals and reagents were purchased from Sigma𠄺ldrich. The sample was prepared as follows. All steps were performed at room temperature long incubations were conducted in a water-saturated container to avoid evaporation of the sample. A solution containing 20 μg/ml anti-digoxigenin (1333089, Roche, IN) in phosphate-buffered saline (PBS) buffer [2.7 mM KCl, 137 mM NaCl, 5.4 mM Na2HPO4 and 1.8 mM KH2PO4 (pH 7.4)] was introduced into the flow chamber and incubated for 20 min to insure optimal surface coating. The excess antibody was then removed by washing the chamber 5𠄷 times with 80 μl of LBB buffer [10 mM Tris–HCl (pH 7.4), 200 mM KCl, 0.1 mM EDTA, 5% (v/v) dimethyl sulfoxide (DMSO), 0.2 mM DTT, 0.1 mg/ml α-casein]. The DNA sample (80 μl at a concentration of � ng/ml in LBB buffer: this DNA concentration was chosen to maximize single DNA tethers as described below) was then introduced in the chamber and incubated for 1 h. Unbound DNA was removed by washing with 7 × 50 μl LBB − buffer (LBB lacking DTT and DMSO). Streptavidin-coated polystyrene microspheres (diameter 440 nm, Indicia Biotechnology, France) in LBB − buffer were then introduced in the flow chamber and incubated for 30 min. Unbound microspheres were finally removed with 5 × 50 μl washes of LBB buffer supplemented with Lac repressor at a tetramer concentration of 4, 20 or 100 pM, depending on the experiment. The flow chamber was then sealed with silicon grease to allow prolonged observation at the microscope (each slide could be observed for several hours without detectable loss in activity of LacI).

    Data collection and analysis

    The slide was mounted on an inverted microscope (Eclipse TE300, Nikon, Japan), images of a region of interest containing the microsphere chosen were acquired at a frequency of 25 Hz with a video camera (C3077-71, Hamamatsu, Japan) and digitized with an A/D converter (IMAQ PCI-1408, National Instruments, TX). The software for instrumentation control, data acquisition and data processing was written in Labview (v. 6.0, National Instruments, TX). Microspheres were chosen for recording based on the range of mobility exhibited each microsphere was normally monitored for 𢏁 h.

    The position of the microsphere in the sample plane (x-y plane) was monitored in real-time during the experiment using a centroid algorithm (24,25): each video frame was deinterlaced and calculation of the centroid was performed separately, after background subtraction, on the even and odd lines of the image, to obtain measurements of the bead's position with a frequency of 50 Hz. The average radius of mobility of the microsphere was calculated in real-time and charted, during the experiment, at the desired frequency. The x i y coordinates of the microsphere, determined at a frequency of 50 Hz, were also stored for subsequent analysis as described below. Symmetry of the distribution of centroid positions in the x-y plane was used as diagnostic to insure that the microsphere would not be tethered by multiple DNA molecules or be perturbed in its diffusive motion by surface irregularities (25,26). Additionally, the DNA dilution adopted for all experiments had been previously optimized for a relatively high yield of tethered microspheres (density of about 1 tethered microsphere in each microscope field of 34 × 26 μm 2 ) with a negligible probability of multiple DNA molecules bound to the same microsphere.

    At the end of the experiment, data were analyzed as follows. Slow apparatus fluctuations and drift in stage position were removed from the x(t) i y(t) recordings by filtering the data with a Butterworth high-pass filter (cutoff frequency of 0.1 Hz). From the filtered data, R(t) was then calculated as [ x ( t ) ] 2 + [ y ( t ) ] 2 and a running average 〈R(t)〉 was calculated by filtering the R(t) trace with a Gaussian filter (27). For each microsphere a set of four 〈R(t)〉 traces was calculated using Gaussian filters with cutoff frequencies of 0.133, 0.066, 0.044 and 0.033 Hz, corresponding to standard deviations of the filter's impulse response of 1, 2, 3 and 4 s, respectively. The left panels of Figures 2 and ​ and3 3 show examples of 〈R(t)〉 traces obtained with a filter cutoff frequency of 0.066 Hz.

    Examples of TPM experimental recordings obtained with wild-type LacI. The left panels show time courses of the average radius of microsphere mobility, 〈R(t)〉 (calculated as described in the Materials and Methods section, choosing a Gaussian filter with cutoff frequency of 0.066 Hz) obtained with LacI at a concentration of 100 pM (a), 20 pM (c), and 4 pM (e). The right panels show the distributions of 〈R(t)〉 corresponding to each of the recordings shown on the left. The lines represent the best fit of the sum of two Gaussians (see Materials and Methods) to the histogram. The fitted parameters are shown in the insets.

    Examples of TPM experimental recordings obtained with LacI mutants Q60G and Q60 + 1. The left panels show time courses of the average radius of mobility, 〈R(t)〉 (calculated as in Figure 2 ), obtained with Q60G (a) and Q60 + 1 (c) at a concentration of 100 pM. Histograms (shown on the right) are fitted with two Gaussians as described in Figure 2 .

    Dwell-times were extracted from each 〈R(t)〉 recording using a half-amplitude threshold method (27): the 〈R(t)〉 distribution (the right panels of Figures 2 and ​ and3 3 show some examples) was fitted with a double Gaussian: A 1 exp [ − ( x − x c 1 ) 2 / 2 σ 1 2 ] + A 2 exp [ − ( x − x c 2 ) 2 / 2 σ 2 2 ] . For each recording, the threshold was set half way between the peaks of the two fitted Gaussian curves.

    A first semi-quantitative analysis of dwell-times was elaborated following a method analogous to that described by Finzi and Gelles (20), which is based on the choice of a single filter: the R(t) data were filtered with cutoff frequency of 0.033 Hz, corresponding to a filter dead time (Td) of 5.4 s. The transitions giving raise to events shorter than Td were ignored (20,28) and the resulting dwell-time distributions were analyzed to produce the durations histograms, reporting only the events with durations longer than 2Td (20,27).

    Then, a full characterization of the analysis method was conducted using filters with different cutoff frequencies (0.133, 0.066, 0.044 and 0.033 Hz) as mentioned above. From the measured distributions of dwell-times, the average lifetime of the looped (τLm) and the unlooped (τUm) states were calculated for each experimental condition studied. In the results section we will show that these measured parameters strongly depend on the choice of filter operated. Thus, we developed a set of mathematical corrections aimed at obtaining, from the TPM measurements, a reliable estimate of the average duration of the looped and unlooped state in the DNA molecule, regardless of the choice of filter and the effects of residual noise in the measurements (see Supplementary Data). For this analysis, dwell-times were measured without imposing any cutoff in duration, since filter's limited time resolution (27) and false events due to noise are taken into account in our analysis method.

    The full description of the derivation of our novel method will be published elsewhere here we provide the mathematical expressions applied to the experimental results presented in this paper for wt-LacI at different concentrations and the Q60G and Q60 + 1 mutants. A description of the corrections used in this work and a demonstration of the validity of this method, based on numerical simulations, is reported in the Supplementary Data.


    Use of Escherichia coli operon-fusion strains for the study of glycerol 3-phosphate transport activity.

    Strains of Escherichia coli K-12 deleted in the native lac operon and bearing both a wild-type glpT operon encoding for sn-glycerol 3-phosphate (G3P) transport and a hybrid operon in which glpT operator and promoter regions are fused to the lacZ gene were constructed. In strains with such a hybrid operon, beta-galactosidase and beta-galactoside permease become inducible by G3P. In these mutants the function and maturation of the glpT-coded proteins should be distinguishable from the level of gene expression, since the beta-galactosidase activity can serve as an index of the latter. With the aid of such mutants, it was shown that: (i) the expressions of the two neighboring operons, glpT and glpA (encoding anaerobic G3P dehydrogenase), are not coordinate (ii) upon induction, the appearance of the cytoplasmic beta-galactosidase activity preceded that of methyl-beta-D-thiogalactoside transport activity (requiring only a cytoplasmic membrane protein) by about 4 min and that of G3P transport activity (requiring both a cytoplasmic membrane protein and a periplasmic protein) by about 9 min and (iii) when cells grown at several temperatures from 24 to 42 degrees C were measured for G3P transport activity at 30 degrees C, the activity increased with the growth temperature, indicating that, within the range studied, the rate of transport increases with the fluidity of membrane phospholipids.


    DISKUSIJA

    Due to its versatility and simplicity, the TPM method has been used for the study of a variety of biochemical systems at the single molecule level ( 20 , 24 , 25 , 33 – 36 ). With regard to dynamic measurements, the main limitation of this experimental approach is represented by the low signal-to-noise ratios that can be accomplished. This limitation is mostly determined by the size of the microspheres used, which defines the time necessary for the diffusive motion to explore the volume available for a certain tether length (see Figure 1 ) variations in the length of the tether can be detected on timescales longer than this characteristic diffusion time. This limitation may be overcome and much higher signal-to-noise ratios (or, equivalently, higher temporal resolution in the measurements) may be accomplished substituting much faster diffusing objects (such as quantum dots) for the microspheres thus far used. When implemented using microspheres, however, TPM requires care in the quantitative interpretation of the measured lifetimes. Figure 7 , in fact, demonstrates how filtering of the experimental recordings (needed to obtain a good discrimination between the loop and unloop states) strongly influences the values of the measured lifetimes. We have elaborated a method of analysis of TPM data to overcome these problems and reliably measure the kinetic parameters of Lac repressor-induced loop formation and disruption.

    The formation of the 305 bp long loop in our DNA construct by simultaneous binding of the Lac repressor tetramer to the two operators is associated with a bending energy in the DNA molecule corresponding to about 9.5 kBT ( 37 , 38 ), calculated simplifying the loop geometry to a circle, and with a DNA persistence length of 50 nm ( 39 , 40 ). The spacing between operators in the Lac operon is 92 and 401 bp ( 4 , 5 ), thus the bending energies involved in the formation of these loops can be significant and may play an important role in the kinetics of transcription regulation. The effects of DNA tension and torsion on the kinetics of DNA-binding proteins have recently been explored in a variety of theoretical studies ( 13 – 15 ) and in experimental measurements on Gal repressor ( 41 ).

    Single molecule approaches are providing fundamental information on the mechanical properties of a growing number of enzymatic systems in vitro ( 42 ). The measurement of forces in the pN range has indicated nucleic acid processing enzymes (such as RNA polymerase, DNA polymerase, topoisomerases) as molecular motors ( 43 – 45 ) capable of producing forces even larger than those of ‘classic’ motors, such as myosin ( 46 ) or kinesin ( 47 , 48 ). These forces may be crucial for these enzymes to overcome obstacles, unwind double-stranded structures and move along the DNA template under the conditions of compaction, tension and torsion to which it is subject in vivo . Transcription regulation systems, including the Lac operon, are sensitive to these forces to the extent by which binding and dissociation of the regulatory proteins are influenced by the bending and twisting energetics of the DNA. Measurements based on the use of magnetic tweezers have clearly demonstrated the sensitivity of the GalR system to supercoiling in the DNA target molecule ( 41 ). The persistence length of DNA in vivo is much lower than that measured in vitro ( 6 ), due to the action of accessory proteins. It is presumable that this physical property of DNA can be modulated to some extent by the quantity and quality of accessory proteins expressed in the cell. Thus, a new concept is recently emerging in gene regulation: the physical properties of DNA may play an important role in shaping the dynamics of gene regulation ( 38 , 49 ).

    We have applied the TPM technique, in combination with a new method for data analysis, to the investigation of the effects of flexibility (both in DNA and in the hinge region of the protein) on the kinetics of loop formation and disruption.

    The analysis of the kinetics of loop disruption is simplified by the fact that there is a 1:1 correspondence between the TPM state of lower microsphere mobility and the biochemical ORO state (see Figure 6 ). This correspondence is confirmed by the lack of dependence of τ Lm on LacI concentration ( Figure 4 , left panels Figure 7a , filled symbols). In fact, as an alternative interpretation, one could imagine a fast equilibrium between O-OR and ORO, characterized by rates much faster then the diffusion rate of the microsphere and shifted toward ORO, so to give raise to an apparent TPM loop state concealing fast biochemical reactions. However, if this were the case, one would expect the durations of the observed TPM loop state to depend on the concentration of LacI. In fact, the exit from the fast equilibrium would be determined by a reaction leading the system into a third, long lived, state. The most relevant long lived state in this regard depends on the concentration of LacI, and thus the entire behavior of the system is dependent on this parameter. More precisely, the ratio between the rates from O-OR toward RO-OR and O-O is given by [LacI]/ KD , which has a value of about 10, 2 and 0.4 for [LacI] of 100, 20 and 4 pM, respectively. At the two higher concentrations, RO-OR is, thus, prevalent: under these conditions, the rate of transition between O-OR and the other main unloop state scales linearly with [LacI]. Thus, the probability of exiting the above mentioned hypothetical equilibrium would also scale linearly with [LacI]. Since the measured duration of the TPM loop state does not depend on the concentration of LacI over a broad range of concentrations [4, 20 and 100 pM tested in this work, and 100 pM and 1 nM tested by Finzi and Gelles ( 20 )] the hypothesis of the TPM loop state underlying a fast biochemical equilibrium is not likely rather, the exit from this TPM state monitors directly the disruption of the loop in the DNA molecule. Thus, the TPM measurement provides a means for directly monitoring the effect of the loop strain on the kinetics of dissociation, as described in the results by the parameter α. Our findings indicate a weak dependence of the rate of loop disruption on the DNA bending and twisting energy, as demonstrated by the value of α between 1 and 2.

    With regard to the kinetics of loop formation, on the other hand, our measurements indicate that formation of a loop in the DNA molecule by binding of Lac repressor simultaneously to two operators is highly sensitive to the DNA bending energy. Qualitatively, this result is well-expected based both on theoretical considerations and previous ligase-catalyzed circularization experiments however, our results on LacI are somewhat surprising quantitatively. In fact, the values we have obtained for Jm (of the order of 10 −10 M) are significantly lower than those measured by ligase-catalyzed cyclization experiments [>10 −8 M for DNA segments of 300 bp ( 8 )] and calculated from physical models of the DNA molecule ( 11 ). It is fundamental to evaluate the effects of the microsphere on the values measured for Jm by TPM technique. It is expected, in fact, that the presence of the microsphere may slow down the kinetics of loop formation, due to an effective swelling force exerted entropically by the microsphere on the polymer. Below we will discuss results indicating that, in a system like the one we setup for the measurements on LacI, the effect of the microsphere on the measured kinetics of loop formation (and, therefore, on the measured Jm ) should not exceed, at most, a factor of 10.

    In their theoretical work, extensively modeling the physical properties of the TPM system, Segall et al . provide an analytical expression [Equation 12 in Ref. ( 50 )] for the swelling force as a function of DNA contour length, persistence length and microsphere radius. In our experimental system, the swelling force estimated according to this expression is about 30 fN. Segall et al . ( 50 ) conclude that a force of this magnitude due to the presence of the microsphere would decrease the rate of looping by about a factor of 2.

    Further, the TPM recordings of the position distributions of the microsphere in the absence of Lac repressor exhibit interesting non-Gaussian features ( 25 ) which may be exploited to estimate the magnitude of the swelling force. Using numerical simulations of the TPM system [similar to those described by Segall et al . ( 50 )] to fit our data, we have estimated in our system an effective swelling force of 127 ± 14 fN (best estimate ± range for 95.4% confidence see Supplementary Data). Recent theoretical works ( 14 , 51 ) have described the effects of force on Jm : a force in the range between 30 and 140 fN due to the presence of the microsphere would not be expected to decrease Jm by more than a factor of 10.

    Finally, Hsieh et al . ( 19 ) reported an estimate of the equilibrium constant K * for the intramolecular looping reaction: for the pRW490 construct containing the two primary operators at a distance of 305 bp from each other, they reported a value of 16 for K * . In our measurements, the equilibrium constant for the intramolecular looping reaction is given by K ′ = Jmka / (2 kd α). Using the values of Jm and α reported in Table 1 we obtain K ′ between 2.5 and 7. The difference between K * i K ′ is to be attributed to the microsphere, according to the following relationship: − RT ln( K ′) = Δ Gtpm = Δ GDNA/LacI + Δ Gbead , gdje je Δ GDNA/LacI = − RT ln( K* ) is the free energy of looping in the absence of microsphere and Δ Gbead is the positive free energy contribution due to the microsphere opposing an entropic force to the formation of the loop. From these relations, based on the measurement of K ′ reported above, we estimate Δ Gbead between 0.8 and 1.8 kBT . If we assume that this energetic barrier affects mostly the rate of formation of the loop, this would lead to a reduction of the looping rate by at most 55–83%, in agreement with what calculated by Segall et al . ( 50 ). Thus, we conclude that, even accounting for an underestimation of Jm by about an order of magnitude in TPM due to the microsphere, Lac repressor looping is characterized by a Jm much (at least 10 times) lower than the ligase-catalyzed circularization of a DNA segment of the same length.

    The shape of a DNA loop can vary over a wide range of geometries determining large variations in the loop energetics and in the resulting Jm values ( 15 , 49 ), with significant deviations from those measured in cyclization experiments. Also, the effect of the protein bridging between the two extremities of the loop significantly influences the calculated values of Jm ( 52 , 53 ).

    The structure of the DNA–LacI complex in the looped configuration has not yet been determined. In addition to the original V-shaped model, proposed by Lewis et al . ( 54 ), an extended conformation of the Lac repressor has been proposed ( 55 ). The rigidity of these conformations and the possibility for the protein to switch between these multiple structural states are fundamental in determining Jm for loop formation. Also, distance and phasing between the operators is a fundamental factor in determining the value of Jm , as already demonstrated for ligase-catalyzed circularization. The TPM method holds promise to allow a systematic characterization of the dependence of LacI regulation of the Lac operon on each of these components, providing in the near future, a complete picture of the orientation effects and of the protein and DNA mechanics involved in the process.

    Our measurements on the mutants of the hinge region Q60G and Q60 + 1 indicate that alterations in the flexibility and geometry of the hinge lead to significant changes in the kinetics measurable with TPM. The association and dissociation rate constants for these mutants have not been measured in standard biochemical assays however, the data shown in Figure 7b clearly demonstrate the large effects of these mutations on the looped lifetimes, with much smaller effects on the unlooped lifetimes. These results lead to the conclusion that the mutations studied cause changes predominantly in the value of the dissociation rate constant. As expected, the mutant characterized by the lower equilibrium dissociation constant (Q60G) displays a longer looped average lifetime, whereas the mutant with the higher KD (Q60 + 1) displays a shorter looped average lifetime. However, the sensitivity of the protein to DNA strain is not significantly affected by these mutations. It is likely that a role of this kind may be more appropriate for the tetramerization domain, and for the regions of interaction between monomers in the tetramer, which affect the propensity of the protein to take a V-shape, an open shape or other possible conformations responsible for a different sensitivity to strain in the DNA target.

    In conclusion, with regard to DNA bending, our work indicates that the rate of loop formation by Lac repressor depends more strongly than previously expected on the energetics of bending and twisting of DNA. Therefore, the mechanical and biochemical factors that in vivo modulate these energetics can play a crucial role in the modulation of gene expression regulation at least in the paradigmatic example of the Lac operon. With regard to the protein flexibility, on the other hand, our results show that the hinge flexibility and geometry have a determinant effect especially on the lifetime of the looped state, demonstrating the interplay of the mechanical properties of partners in this classic example of protein–DNA interacting system.

    Future steps aimed at a further characterization of the TPM system will investigate the effect of microspheres of different sizes on TPM measurements. Also, the measurement of looping and unlooping kinetics of different constructs in which the distances and phasing between operators is varied will provide important additional information.


    Pogledajte video: The Lac operon. Regulation of gene expression (Februar 2023).