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SS1_2018_Lesing_11 - Biologie

SS1_2018_Lesing_11 - Biologie



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Proteïensintese

Inleiding

Die proses van vertaling in biologie is die dekodering van 'n mRNA-boodskap in 'n polipeptiedproduk. Anders gestel, 'n boodskap wat in die chemiese taal van nukleotiede geskryf is, word in die chemiese taal van aminosure "vertaal". Aminosure word lineêr saamgespan via kovalente bindings (genoem peptiedbindings) tussen amino- en karboksieltermini van aangrensende aminosure. Die dekodering en "koppeling" proses word gekataliseer deur 'n ribonukleoproteïen kompleks genaamd die ribosome en kan lei tot kettings van aminosure van lengtes wat wissel van tiene tot meer as 1 000.

Die gevolglike proteïene is so belangrik vir die sel dat hul sintese meer van 'n sel se energie verbruik as enige ander metaboliese proses. Soos DNA-replikasie en transkripsie, is vertaling 'n komplekse molekulêre proses wat ons kan benader deur beide die Energy Story- en Design Challenge-rubrieke te gebruik. Die beskrywing van die algehele proses, of stappe in die proses, vereis die boekhouding van die materie en energie voor die proses en na die proses en 'n beskrywing van hoe daardie materie getransformeer en energie oorgedra word tydens die proses. Vanuit 'n Ontwerpuitdaging-oogpunt kan ons - selfs voordat ons verder delf na wat oor vertaling verstaan ​​word of nie - probeer om van die basiese vrae af te lei wat ons oor hierdie proses sal moet beantwoord.

Kom ons begin deur die basiese probleem te oorweeg. Ons het 'n string RNA (genoem mRNA) en 'n klomp aminosure en ons moet op een of ander manier 'n masjien ontwerp wat:

(a) dekodeer die chemiese taal van nukleotiede in die taal van aminosure,
(b) verbind aminosure op 'n baie spesifieke wyse,
(c) hierdie proses met redelike akkuraatheid voltooi, en
(d) doen dit teen 'n redelike spoed. Redelik, word natuurlik gedefinieer deur natuurlike seleksie.

Soos voorheen kan ons subprobleme identifiseer

(a) Hoe bepaal ons molekulêre masjien waar en wanneer om te begin werk?
(b) Hoe koördineer die molekulêre masjien dekodering en bindingsformasies?
(c) waar kom die energie vir hierdie proses vandaan en hoeveel?
(d) hoe weet die masjien waar om te stop?

Ander vrae en funksionele probleme/uitdagings sal beslis ontstaan ​​soos ons dieper delf.

Die punt, soos altyd, is dat selfs sonder om enige besonderhede oor vertaling te weet, ons ons verbeelding, nuuskierigheid en gesonde verstand kan gebruik om 'n paar vereistes vir die proses voor te stel waaroor ons meer sal moet leer. Om hierdie vrae te verstaan ​​as die konteks vir wat volg, is die sleutel.

'n Peptiedbinding verbind die karboksielkant van een aminosuur met die amino-einde van 'n ander, wat een watermolekule verdryf. Die R1 en R2 benaming verwys na syketting van aminosuur die twee aminosure.
Erkenning: Marc T. Facciotti (oorspronklike werk).

Masjinerie vir proteïensintese

Die komponente wat in die proses ingaan

Baie verskillende molekules en makromolekules dra by tot die proses van translasie. Terwyl die presiese samestelling van "die spelers" in die proses van spesie tot spesie kan verskil - kan ribosome byvoorbeeld uit verskillende getalle bestaan rRNA's (ribosomale RNA's) en polipeptiede afhangende van die organisme - die algemene funksies van die proteïensintese-masjinerie is vergelykbaar van bakterieë tot menslike selle. Ons fokus op hierdie ooreenkomste. Op 'n minimum vereis vertaling 'n mRNA-sjabloon, aminosure, ribosome, tRNA's, 'n energiebron, en verskeie bykomende bykomstige ensieme en klein molekules.

Herinnering: Aminosure

Kom ons onthou eenvoudig dat die basiese struktuur van aminosure saamgestel is uit 'n ruggraat wat saamgestel is uit 'n aminogroep, 'n sentrale koolstof (genoem die α-koolstof) en 'n karboksielgroep. Aan die α-koolstof is 'n veranderlike groep geheg wat help om van die chemiese eienskappe en reaktiwiteit van die aminosuur te bepaal.

'n Generiese aminosuur.
Erkenning: Marc T. Facciotti (eie werk)

Die 20 algemene aminosure.
Erkenning: Marc T. Facciotti (eie werk)

Ribosome

A ribosoom is 'n komplekse makromolekule wat bestaan ​​uit strukturele en katalitiese rRNA's, en baie afsonderlike polipeptiede. Soos ons begin dink aan energierekening in die sel is dit opmerklik dat ribosome nie "vry" kom nie. Selfs voordat 'n mRNA vertaal word, moet 'n sel energie belê om elkeen van sy ribosome te bou. In E coli, is daar tussen 10 000 en 70 000 ribosome teenwoordig in elke sel op enige gegewe tydstip.

Ribosome bestaan ​​in die sitoplasma in bakterieë en archaea en in die sitoplasma en op die growwe endoplasmiese retikulum in eukariote. Mitochondria en chloroplaste het ook hul eie ribosome in die matriks en stroma, wat meer soortgelyk aan bakteriële ribosome lyk (en soortgelyke geneesmiddelsensitiwiteit het), as die ribosome net buite hul buitenste membrane in die sitoplasma. Ribosome dissosieer in groot en klein subeenhede wanneer hulle nie proteïene sintetiseer nie en herassosieer tydens die aanvang van translasie. coli, word die klein subeenheid beskryf as 30S, en die groot subeenheid is 50S. Soogdierribosome het 'n klein 40S subeenheid en 'n groot 60S subeenheid. Die klein subeenheid is verantwoordelik vir die binding van die mRNA-sjabloon, terwyl die groot subeenheid tRNA's opeenvolgend bind. Elke mRNA-molekule word gelyktydig deur baie ribosome vertaal, almal sintetiseer proteïen in dieselfde rigting: lees die mRNA van 5' tot 3' en sintetiseer die polipeptied vanaf die N-terminus na die C-terminus. Die volledige mRNA/poli-ribosoomstruktuur word a genoem polisoom.

Die proteïensintese-masjinerie sluit die groot en klein subeenhede van die ribosoom, mRNA en tRNA in.
Bron: http://bio1151.nicerweb.com/Locked/m.../ribosome.html

TRNA's

tRNA's is strukturele RNA-molekules wat vanaf gene getranskribeer is. Afhangende van die spesie, bestaan ​​40 tot 60 tipes tRNA's in die sitoplasma. Dien as adapters, spesifieke tRNA's bind aan volgordes op die mRNA-sjabloon en voeg die ooreenstemmende aminosuur by die polipeptiedketting. Daarom is tRNA's die molekules wat eintlik die taal van RNA "vertaal" in die taal van proteïene.

Van die 64 moontlike mRNA kodons—of drielingkombinasies van A, U, G en C, drie spesifiseer die beëindiging van proteïensintese en 61 spesifiseer die byvoeging van aminosure tot die polipeptiedketting. Van hierdie 61 kodeer een kodon (AUG) ook die aanvang van translasie. Elke tRNA antikodon kan basispaar met een van die mRNA-kodons en 'n aminosuur byvoeg of vertaling beëindig, volgens die genetiese kode. Byvoorbeeld, as die volgorde CUA op 'n mRNA-sjabloon in die regte leesraam voorkom, sal dit 'n tRNA bind wat die komplementêre volgorde, GAU, uitdruk wat aan die aminosuur leucine gekoppel sal wees.

Die gevoude sekondêre struktuur van 'n tRNA. Die antikodonlus en aminosuur-aanvaarderstam word aangedui.
Bron: http://mol-biol4masters.masters.grkr...ansfer_RNA.htm

Aminoasiel tRNA-sintetases

Die proses van pre-tRNA-sintese deur RNA-polimerase III skep slegs die RNA-gedeelte van die adaptermolekule. Die ooreenstemmende aminosuur moet later bygevoeg word, sodra die tRNA verwerk en na die sitoplasma uitgevoer is. Deur die proses van tRNA "laai" word elke tRNA-molekule aan sy korrekte aminosuur gekoppel deur 'n groep ensieme genaamd aminoasiel tRNA sintetases. Ten minste een tipe aminoasiel tRNA sintetase bestaan ​​vir elk van die 20 aminosure; die presiese aantal aminoasiel-tRNA-sintetases verskil volgens spesie. Hierdie ensieme bind en hidroliseer eers ATP om 'n hoë-energiebinding tussen 'n aminosuur en adenosienmonofosfaat (AMP) te kataliseer; 'n pirofosfaatmolekule word in hierdie reaksie uitgedryf. Die geaktiveerde aminosuur word dan na die tRNA oorgedra, en AMP word vrygestel.

Die meganisme van proteïensintese

Net soos met mRNA-sintese, kan proteïensintese in drie fases verdeel word: aanvang, verlenging en beëindiging. Die proses van translasie is soortgelyk in bakterieë, archaea en eukariote.

Vertaling inisiëring

Oor die algemeen begin proteïensintese met die vorming van 'n inisiasiekompleks. Die klein ribosomale subeenheid sal aan die mRNA bind by die ribosomale bindingsplek. Kort daarna sal die metionien-tRNA aan die AUG-beginkodon bind (deur komplementêre binding met sy antikodon). Hierdie kompleks word dan verbind deur 'n groot ribosomale subeenheid. Hierdie inisiasiekompleks werf dan die tweede tRNA en dus begin translasie.

Translasie begin wanneer 'n tRNA-antikodon 'n kodon op die mRNA herken. Die groot ribosomale subeenheid sluit aan by die klein subeenheid, en 'n tweede tRNA word gewerf. Soos die mRNA relatief tot die ribosoom beweeg, word die polipeptiedketting gevorm. Invoer van 'n vrystellingsfaktor in die A-werf beëindig vertaling en die komponente dissosieer.

Bakteriële vs eukariotiese inisiasie

In E coli mRNA, 'n volgorde stroomop van die eerste AUG-kodon, genoem die Shine-Dalgarno-volgorde (AGGAGG), interaksie met 'n rRNA-molekule. Hierdie interaksie anker die 30S ribosomale subeenheid op die korrekte plek op die mRNA-sjabloon. Stop vir 'n oomblik om die herhaling van 'n meganisme wat jy al teëgekom het te waardeer. In hierdie geval, om 'n proteïenkompleks te kry om - in behoorlike register - met 'n nukleïensuurpolimeer te assosieer, word bewerkstellig deur twee antiparallelle stringe komplementêre nukleotiede met mekaar in lyn te bring. Ons het dit ook gesien in die funksie van telomerase.

In plaas daarvan om by die Shine-Dalgarno-volgorde te bind, herken die eukariotiese inisiasiekompleks die 7-metielguanosienkap aan die 5'-kant van die mRNA. 'n Kapbindende proteïen (CBP) help die beweging van die ribosoom na die 5'-dop. Sodra dit by die pet is, volg die inisiasiekompleks langs die mRNA in die 5'- tot 3'-rigting, op soek na die AUG-beginkodon. Baie eukariotiese mRNA's word vanaf die eerste AUG vertaal, maar dit is nie altyd die geval nie. Volgens Kozak se reëls, dui die nukleotiede rondom die AUG aan of dit die korrekte beginkodon is. Kozak se reëls bepaal dat die volgende konsensusvolgorde rondom die AUG van gewerwelde gene moet verskyn: 5'-gccRccAUGG-3'. Die R (vir purien) dui 'n plek aan wat óf A óf G kan wees, maar nie C of U kan wees nie. In wese, hoe nader die volgorde aan hierdie konsensus is, hoe hoër is die doeltreffendheid van translasie.

Vertaling verlenging

Tydens translasie-verlenging verskaf die mRNA-sjabloon spesifisiteit. Soos die ribosoom langs die mRNA beweeg, kom elke mRNA-kodon in 'sig', en spesifieke binding met die ooreenstemmende gelaaide tRNA-antikodon word verseker. As mRNA nie in die verlengingskompleks teenwoordig was nie, sou die ribosoom tRNA's nie-spesifiek bind. Let weer op die gebruik van basisparing tussen twee antiparallelle stringe van komplementêre nukleotiede om ons molekulêre masjien in register te bring en te hou en in hierdie geval ook om die werk van "vertaal" tussen die taal van nukleotiede en aminosure te bereik.

Die groot ribosomale subeenheid bestaan ​​uit drie kompartemente: die A-plek bind inkomende gelaaide tRNA's (tRNA's met hul aangehegte spesifieke aminosure), die P-plek bind gelaaide tRNA's wat aminosure dra wat bindings met die groeiende polipeptiedketting gevorm het, maar nog nie gedissosieer het van hul ooreenstemmende tRNA, en die E-plek wat gedissosieerde tRNA's vrystel sodat hulle met 'n ander vrye aminosuur herlaai kan word.

Verlenging gaan voort met gelaaide tRNA's wat die A-plek binnegaan en dan na die P-plek verskuif, gevolg deur die E-plek met elke enkelkodon-“stap” van die ribosoom. Ribosomale stappe word geïnduseer deur konformasieveranderinge wat die ribosoom met drie basisse in die 3'-rigting bevorder. Die energie vir elke stap van die ribosoom word geskenk deur 'n verlengingsfaktor wat GTP hidroliseer. Peptiedbindings vorm tussen die aminogroep van die aminosuur wat aan die A-plek tRNA geheg is en die karboksielgroep van die aminosuur wat aan die P-plek tRNA geheg is. Die vorming van elke peptiedbinding word gekataliseer deur peptidieltransferase, 'n RNA-gebaseerde ensiem wat in die 50S ribosomale subeenheid geïntegreer is. Die energie vir elke peptiedbindingsvorming word verkry van GTP-hidrolise, wat deur 'n aparte verlengingsfaktor gekataliseer word. Die aminosuur gebind aan die P-plek tRNA is ook gekoppel aan die groeiende polipeptiedketting. Soos die ribosoom oor die mRNA stap, gaan die voormalige P-plek tRNA die E-plek binne, los van die aminosuur en word uitgedryf. Die ribosoom beweeg langs die mRNA, een kodon op 'n slag, en kataliseer elke proses wat in die drie terreine plaasvind. Met elke stap gaan 'n gelaaide tRNA die kompleks binne, die polipeptied word een aminosuur langer, en 'n ongelaaide tRNA vertrek. Verbasend genoeg vind hierdie proses vinnig plaas in die sel, die E coli translasieapparaat neem slegs 0,05 sekondes om elke aminosuur by te voeg, wat beteken dat 'n 200-aminosuur polipeptied in net 10 sekondes vertaal kan word.

Voorgestelde bespreking

Baie antibiotika inhibeer bakteriële proteïensintese. Byvoorbeeld, tetrasiklien blokkeer die A-plek op die bakteriële ribosoom, en chlooramfenikol blokkeer peptidieloordrag. Watter spesifieke effek sou jy verwag om elkeen van hierdie antibiotika op proteïensintese te hê?

Die Genetiese Kode

Om op te som wat ons tot op hierdie punt weet, genereer die sellulêre proses van transkripsie boodskapper-RNA (mRNA), 'n mobiele molekulêre kopie van een of meer gene met 'n alfabet van A, C, G en uracil (U). Vertaling van die mRNA-sjabloon omskep nukleotied-gebaseerde genetiese inligting in 'n proteïenproduk. Proteïenvolgordes bestaan ​​uit 20 algemeen voorkomende aminosure; daarom kan gesê word dat die proteïen-alfabet uit 20 letters bestaan. Elke aminosuur word gedefinieer deur 'n drie-nukleotiedvolgorde wat die drieling genoem word kodon. Die verwantskap tussen 'n nukleotiedkodon en sy ooreenstemmende aminosuur word die genoem genetiese kode. Gegewe die verskillende getalle van "letters" in die mRNA en proteïen "alfabette," beteken dat daar 'n totaal van 64 (4 × 4 × 4) moontlike kodons; daarom moet 'n gegewe aminosuur (20 totaal) deur meer as een kodon gekodeer word.

Drie van die 64 kodons beëindig proteïensintese en stel die polipeptied vry van die translasiemasjinerie. Hierdie drieling word genoem stop kodons. 'n Ander kodon, AUG, het ook 'n spesiale funksie. Benewens die spesifiseer van die aminosuur metionien, dien dit ook as die begin kodon vertaling te begin. Die leesraam vir translasie word gestel deur die AUG-beginkodon naby die 5'-kant van die mRNA. Die genetiese kode is universeel. Met 'n paar uitsonderings, gebruik feitlik alle spesies dieselfde genetiese kode vir proteïensintese, wat 'n kragtige bewys is dat alle lewe op Aarde 'n gemeenskaplike oorsprong deel.

Hierdie figuur toon die genetiese kode vir die vertaling van elke nukleotiedtriplet, of kodon, in mRNA in 'n aminosuur of 'n terminasiesein in 'n ontluikende proteïen. (krediet: wysiging van werk deur NIH)
Oorbodig, nie dubbelsinnig nie

Die inligting in die genetiese kode is oorbodig. Veelvuldige kodons kodeer vir dieselfde aminosuur. Byvoorbeeld, deur die grafiek hierbo te gebruik, kan jy 4 verskillende kodons vind wat vir Valine kodeer, net so is daar twee kodons wat vir Leucine kodeer, ens. Maar die kode is nie dubbelsinnig nie, wat beteken dat as jy 'n kodon gegee word, jy sou definitief weet vir watter aminosuur dit kodeer, sal 'n kodon slegs vir 'n spesifieke aminosuur kodeer. Byvoorbeeld, GUU sal altyd vir Valine kodeer, en AUG sal altyd vir Methionine kodeer. Dit is belangrik, jy sal gevra word om 'n mRNA in 'n proteïen te vertaal deur 'n kodonkaart soos die een hierbo getoon te gebruik.

Vertaling beëindiging

Beëindiging van translasie vind plaas wanneer 'n stopkodon (UAA, UAG of UGA) teëgekom word. Wanneer die ribosoom die stopkodon teëkom, kom geen tRNA in die A-plek binne nie. In plaas daarvan 'n proteïen bekend as 'n vrystelling faktor bind aan die kompleks. Hierdie interaksie destabiliseer die translasiemasjinerie, wat die vrystelling van die polipeptied en die dissosiasie van die ribosoomsubeenhede van die mRNA veroorsaak. Nadat baie ribosome translasie voltooi het, word die mRNA afgebreek sodat die nukleotiede in 'n ander transkripsie-reaksie hergebruik kan word.

Voorgestelde bespreking

Wat is die voordele en nadele daaraan verbonde om 'n enkele mRNA verskeie kere te vertaal?

Koppeling tussen transkripsie en vertaling

Soos voorheen bespreek, hoef bakterieë en archaea nie hul RNA-transkripsies tussen 'n membraangebonde kern en die sitoplasma te vervoer nie. Die RNA-polimerase is dus besig om RNA direk in die sitoplasma te transkribeer. Hier kan ribosome aan die RNA bind en die proses van translasie begin, in sommige gevalle terwyl transsipsie nog plaasvind. Die koppeling van hierdie twee prosesse, en selfs mRNA-afbraak, word nie net vergemaklik omdat transkripsie en translasie in dieselfde kompartement plaasvind nie, maar ook omdat beide prosesse in dieselfde rigting plaasvind - sintese van die RNA-transkripsie vind in die 5' tot 3 plaas. ' rigting en vertaling lees die transkripsie in die 5' tot 3' rigting. Hierdie "koppeling" van transkripsie met vertaling vind plaas in beide bakterieë en archaea en is in werklikheid noodsaaklik vir behoorlike geenuitdrukking in sommige gevalle.

Veelvuldige polimerases kan 'n enkele bakteriese geen transkribeer terwyl talle ribosome gelyktydig die mRNA-transkripsies in polipeptiede vertaal. Op hierdie manier kan 'n spesifieke proteïen vinnig 'n hoë konsentrasie in die bakteriese sel bereik.

Proteïen sortering

In konteks van 'n proteïensintese-ontwerpuitdaging kan ons ook die vraag/probleem opper van hoe proteïene kom waar hulle veronderstel is om te gaan. Ons weet dat sommige proteïene bestem is vir die plasmamembraan, ander in eukariotiese selle moet na verskeie organelle gerig word, sommige proteïene, soos hormone of proteïene wat voedingstowwe opvang, is bedoel om deur selle afgeskei te word terwyl ander na dele gerig moet word. van die sitosol om strukturele rolle te dien. Hoe gebeur dit?

Aangesien verskeie meganismes ontbloot is, word die besonderhede van hierdie proses nie maklik in 'n kort paragraaf of twee opgesom nie. Enkele sleutel-gemeenskaplike elemente van alle meganismes kan egter genoem word. Eerstens is die behoefte aan 'n spesifieke "merker" wat 'n bietjie molekulêre inligting kan verskaf oor waar die proteïen van belang bestem is. Hierdie merker neem gewoonlik die vorm aan van 'n kort string aminosure - 'n sogenaamde seinpeptied - wat inligting kan kodeer oor waar die proteïen bedoel is om te eindig. Die tweede vereiste komponent van die proteïensorteermasjinerie moet 'n stelsel wees om die proteïene werklik te lees en te sorteer. In bakteriële en argaeale sisteme bestaan ​​dit gewoonlik uit proteïene wat die seinpeptied tydens translasie kan identifiseer, daaraan kan bind en die sintese van die ontluikende proteïen na die plasmamembraan kan lei. In eukariotiese stelsels is die sortering noodwendig meer kompleks, en behels 'n taamlik uitgebreide stel meganismes van seinherkenning, proteïenmodifikasie en handel in vesikels tussen organelle of die membraan. Hierdie biochemiese stappe word in die endoplasmiese retikulum geïnisieer en verder "verfyn" in die Golgi-apparaat waar proteïene gemodifiseer en verpak word in vesikels wat vir verskeie dele van die sel gebind word.

Sommige van die verskillende spesifieke meganismes kan deur jou instrukteur in die klas bespreek word. Die sleutel vir alle studente dit so waardeer die probleem en om 'n algemene idee te hê van die hoëvlak vereistes wat selle aangeneem het om dit op te los.

Post-translasionele Proteïenmodifikasie

Na translasie kan individuele aminosure chemies gemodifiseer word. Hierdie modifikasies voeg chemiese variasie en nuwe eienskappe by wat gewortel is in die chemie van die funksionele groepe wat bygevoeg word. Algemene modifikasies sluit in fosfaatgroepe, metiel-, asetaat- en amiedgroepe. Sommige proteïene, tipies gerig op membrane, sal gelipideer word - 'n lipied sal bygevoeg word. Ander proteïene sal geglikosileer word - 'n suiker sal bygevoeg word. Nog 'n algemene post-translasionele modifikasie is splitsing of koppeling van dele van die proteïen self. Seinpeptiede kan gesplit word, dele kan uit die middel van die proteïen uitgesny word, of nuwe kovalente koppelings kan tussen sisteïen of ander aminosuur-sykettings gemaak word. Byna alle modifikasies sal deur ensieme gekataliseer word en almal verander die funksionele gedrag van die proteïen.

Afdeling Opsomming

mRNA word gebruik om proteïene te sintetiseer deur die proses van translasie. Die genetiese kode is die ooreenstemming tussen die drie-nukleotied mRNA kodon en 'n aminosuur. Die genetiese kode word "vertaal" deur die tRNA-molekules, wat 'n spesifieke kodon met 'n spesifieke aminosuur assosieer. Die genetiese kode is gedegenereer omdat 64 tripletkodons in mRNA slegs 20 aminosure en drie stopkodons spesifiseer. Dit beteken dat meer as een kodon ooreenstem met 'n aminosuur. Byna elke spesie op die planeet gebruik dieselfde genetiese kode.


Die spelers in vertaling sluit die mRNA-sjabloon, ribosome, tRNA's en verskeie ensiematiese faktore in. Die klein ribosomale subeenheid bind aan die mRNA-sjabloon. Vertaling begin by die aanvang van AUG op die mRNA. Die vorming van bindings vind plaas tussen opeenvolgende aminosure gespesifiseer deur die mRNA-sjabloon volgens die genetiese kode. Die ribosoom aanvaar gelaaide tRNA's, en soos dit langs die mRNA stap, kataliseer dit binding tussen die nuwe aminosuur en die einde van die groeiende polipeptied. Die hele mRNA word in drie-nukleotied-“stappe” van die ribosoom vertaal. Wanneer 'n stopkodon teëgekom word, bind en dissosieer 'n vrystellingsfaktor die komponente en maak die nuwe proteïen vry.

Inleiding tot geenregulering

Regulering gaan alles oor besluitneming. Geenregulering gaan dus alles daaroor om te verstaan ​​hoe selle besluite neem oor watter gene om aan te skakel, af te skakel of om op of af te stem. In die volgende afdeling bespreek ons ​​sommige van die fundamentele meganismes en beginsels wat deur selle gebruik word om geenuitdrukking te reguleer in reaksie op veranderinge in sellulêre of eksterne faktore. Hierdie biologie is belangrik om te verstaan ​​hoe selle veranderende omgewings aanpas, insluitend hoe sommige selle, in meersellige organismes, besluit om gespesialiseerd te word vir sekere funksies (bv. weefsels).

Aangesien die onderwerp van regulering beide 'n baie diep en breë onderwerp van studie in biologie is, probeer ons in Bis2a nie elke detail dek nie - daar is eenvoudig te veel. Eerder, soos ons vir alle ander onderwerpe gedoen het, probeer ons om te fokus op (a) die uiteensetting van sommige van die kern logiese konstrukte en vrae wat jy moet hê wanneer jy ENIGE scenario wat regulering behels, (b) aanleer van 'n paar algemene woordeskat en alomteenwoordige meganismes en (c) 'n paar konkrete voorbeelde te ondersoek wat die punte wat in a en b gemaak is, illustreer.

Geen uitdrukking

Inleiding

Alle selle beheer wanneer en hoeveel elkeen van sy gene uitgedruk word. Hierdie eenvoudige stelling - een wat bloot afgelei kan word van die waarneming van sellulêre gedrag - bring baie vrae na vore wat ons kan begin uitlê deur ons Design Challenge-rubriek te gebruik.

Probeer om "geenuitdrukking" te definieer

Die eerste ding wat ons egter moet doen, is om te definieer wat dit beteken as ons sê dat 'n geen "uitgedruk word". As die geen 'n proteïen kodeer, kan 'n mens redelikerwys voorstel dat "uitdrukking" van 'n geen beteken hoeveel funksionele proteïen gemaak word. Maar wat as die geen nie 'n proteïen kodeer nie, maar eerder 'n paar funksionele RNA. Dan, in hierdie geval, "uitgedruk kan beteken hoeveel van die funksionele RNA gemaak word. Nog 'n ander persoon kan redelikerwys voorstel dat "uitdrukking" net verwys na die aanvanklike stap in die skep van 'n kopie van die genomiese inligting. Volgens daardie definisie kan 'n mens dalk wil tel hoeveel vollengte transkripsies gemaak word.Is dit die aantal eindprodukte wat deur die genomiese inligting gekodeer word of is dit die aantal leeswerk van die inligting wat belangrik is om "uitdrukking" behoorlik te beskryf. Ongelukkig het ons in die praktyk ons vind dikwels dat die definisie afhang van die konteks van die bespreking. Hou dit in gedagte. Om seker te maak dat ons oor dieselfde ding praat, sal ons in Bis2A probeer om die term "uitdrukking" hoofsaaklik te gebruik om die skepping van die finale funksionele produk(te) Afhangende van die spesifieke geval, kan die finale produk 'n proteïen- of RNA-spesie wees.

Die ontwerpuitdaging om geenuitdrukking te reguleer

Om hierdie bespreking vanuit 'n ontwerpuitdagingsperspektief te dryf, kan ons formeel bepaal dat die "groot probleem" waarin ons belangstel om te onderskat, dié is om proteïenoorvloed in 'n sel te reguleer. Probleem: Die oorvloed van elke funksionele proteïen moet gereguleer word. Ons kan dan begin deur subprobleme te stel:

Kom ons neem egter eers 'n oomblik om 'n paar idees te herlaai. Die proses van geenuitdrukking vereis veelvuldige stappe, afhangende van wat die lot van die finale produk sal wees. In die geval van strukturele en regulatoriese RNA's (d.w.s. tRNA, rRNA, snRNA, ens.) vereis die proses dat 'n geen getranskribeer word en dat enige nodige post-transkripsionele prosessering plaasvind. In die geval van 'n proteïenkoderende geen, moet die transkripsie ook in proteïen vertaal word en indien nodig, moet modifikasies aan die proteïen ook gemaak word. Natuurlik is beide transkripsie en vertaling multi-stap prosesse en die meeste van daardie sub-stappe is ook potensiële terreine van beheer.

Sommige van die subprobleme kan dus wees:

  1. Dit is redelik om te postuleer dat daar een of ander meganisme(s) moet wees om die eerste stap van hierdie multi-stap proses te reguleer, die aanvang van transkripsie (net om dinge aan die gang te kry). Dus, kan ons sê, "ons het 'n meganisme nodig om die aanvang van transkripsie te reguleer." Ons kan dit ook omskep in 'n vraag en vra, "hoe kan die aanvang van transkripsie bewerkstellig word"?
  2. Ons kan soortgelyke denke gebruik om te sê, "ons het 'n meganisme nodig om die einde van transkripsie te reguleer" of om te vra "hoe word transkripsie beëindig?"
  3. Deur hierdie konvensie te gebruik, kan ons sê, "ons moet die aanvang van vertaling en die stop van vertaling reguleer".
  4. Ons het net oor sintese van proteïen en RNA gepraat. Dit is redelik om ook te sê, "ons het 'n meganisme nodig om die afbraak van RNA en proteïen te reguleer."

Fokus op transkripsie

In hierdie kursus begin ons deur hoofsaaklik te fokus op die ondersoek van die eerste paar probleme/vrae, die regulering van transkripsie-inisiasie en -terminasie - van genomiese inligting tot 'n funksionele RNA, hetsy gereed soos dit is (bv. in die geval van 'n funksionele RNA) of gereed vir vertaling. Dit stel ons in staat om 'n paar fundamentele konsepte rakende die regulering van geenuitdrukking te ondersoek en om 'n paar werklike voorbeelde van daardie konsepte in aksie te ondersoek.

Voorgestelde bespreking

Hoekom is dit belangrik om geenuitdrukking te reguleer, hoekom nie net alle gene heeltyd uitdruk nie?

Skep 'n lys van hipoteses saam met jou klasmaats van redes waarom die regulering van geenuitdrukking belangrik is vir bakterieë en archaea en vir eukariote. In plaas van bakterieë teen eukariote, wil jy dalk ook kontrasterende redes oorweeg waarom geenregulering belangrik is vir eensellige organismes teenoor veelsellige organismes of gemeenskappe van eensellige organismes (soos kolonies van bakterieë).

Subprobleme vir transkripsie en die aktiwiteit of RNA-polimerase

Kom ons kyk na 'n proteïenkoderende geen en werk deur 'n paar logika. Ons begin deur 'n eenvoudige geval voor te stel, waar 'n proteïenkoderende geen deur 'n enkele aaneenlopende stuk DNA gekodeer word. Ons weet dat om hierdie geen te transkribeer, sal 'n RNA-polimerase gewerf moet word na die begin van die koderende gebied. Die RNA-polimerase is nie "slim" nie op sigself. Daar moet een of ander meganisme wees, gebaseer op chemiese logika, om die RNA-polimerase te help werf na die begin van die proteïenkoderende geen. Net so, as hierdie proses gereguleer moet word, moet daar 'n meganisme of meganismes wees om te bepaal wanneer 'n RNA-polimerase na die begin van 'n geen gewerf moet word, wanneer dit behoort nie, en/of as dit word na die DNA gewerf of dit eintlik met transkripsie moet begin en hoeveel keer hierdie proses behoort te gebeur. Let daarop dat die vorige sin verskeie afsonderlike subprobleme/vrae het (bv. wanneer word die polimerase gewerf?; indien gewerf, moet dit transkripsie begin?; as dit met transkripsie begin, hoeveel keer moet hierdie proses herhaal word?). Ons kan ook redelikerwys aflei dat daar 'n paar meganismes sal moet wees om die polimerase te "opdrag" (meer antropomorfismes) om transkripsie te stop. Ten slotte, aangesien die rol van transkripsie is om RNA-kopieë van die genoomsegmente te skep, moet ons ook probleme/vrae oorweeg wat verband hou met ander faktore wat die oorvloed van RNA beïnvloed, soos meganismes van degradasie. Daar moet sekere meganismes wees en dit sal waarskynlik by die regulering van hierdie proses betrokke wees.

'n Skema wat 'n proteïenkoderende geen toon en sommige van die vrae of probleme wat ons onsself moet vra of alternatiewelik probleme waarvoor ons oplossings moet weet as ons wil verstaan ​​hoe regulering van die transkripsionele gedeelte van die geen se uitdrukking gereguleer word. Erkenning: Marc T. Facciotti (eie werk)

Aktivering en onderdrukking van transkripsie

Sommige basiese beginsels

Kom ons kyk na 'n proteïenkoderende geen en werk deur 'n paar logika. Daar moet een of ander meganisme wees, gebaseer op chemiese logika, om die RNA-polimerase te help werf na die begin van die proteïenkoderende geen. Net so, as hierdie proses gereguleer moet word, moet daar een of ander meganisme, of meganismes, wees om te bepaal wanneer 'n RNA-polimerase na die begin van 'n geen gewerf moet word, wanneer dit nie moet nie, en/of as dit gewerf word na die DNA, of dit eintlik met transkripsie moet begin of nie en hoeveel keer hierdie proses moet gebeur. wanneer word die polimerase gewerf?, indien gewerf moet dit transkripsie begin?, as dit begin transkripsie, hoeveel keer moet hierdie proses herhaal?). Ons kan ook redelikerwys aflei dat daar 'n paar meganismes sal moet wees om die polimerase te "opdrag" (meer antropomorfismes) om transkripsie te stop.

Werwing van RNA-polimerase na spesifieke plekke

Om transkripsie te inisieer, moet die RNA-polimerase gewerf word na 'n segment van DNS naby die begin van 'n DNS-gebied wat vir 'n funksionele transkripsie kodeer. Die funksie van die RNA-polimerase soos tot dusver beskryf, is egter nie om spesifieke volgordes te bind nie, maar eerder om langs enige segment van DNS te beweeg. Om 'n manier te vind om die polimerase na 'n spesifieke terrein te werf, lyk dus teenstrydig met sy gewone gedrag. Om hierdie teenstrydigheid te verduidelik, vereis dat ons iets nuuts aanroep. Enige transkripsie kan enige plek begin en net daardie gebeurtenisse wat lei tot 'n volle produktiewe transkripsie doen enigiets nuttig of iets anders as die RNA-polimerase self help om die ensiem na die begin van 'n geen te werf. Laasgenoemde, aanvaar ons nou as vanselfsprekend, is inderdaad die geval.

Die werwing van die RNA-polimerase word bemiddel deur proteïene wat algemene transkripsiefaktore genoem word. By bakterieë het hulle 'n spesiale naam: sigmafaktore. In archaea word hulle TATA-bindende proteïen en transkripsiefaktor IIB genoem. In eukariote funksioneer familielede van die argaeale proteïene saam met talle ander om die RNA-polimerase te werf. Die algemene transkripsiefaktore het ten minste twee basiese funksies: (1) Hulle is in staat om 'n spesifieke volgorde van DNA chemies te herken en (2) hulle is in staat om die RNA-polimerase te bind. Saam los hierdie twee funksies van algemene transkripsiefaktore die probleem op om 'n ensiem te werf wat andersins nie in staat is om 'n spesifieke DNS-plek te bind nie. In sommige tekste word gesê dat die algemene transkripsiefaktore (en veral die sigmafaktorvariëteite) deel van die RNA-polimerase is. While they are certainly part of the complex when they help to target the RNA polymerase they do not continue with the RNA polymerase after it starts transcription.

The DNA site to which an RNA polymerase is recruited has a special name. It is called a promoter. While the DNA sequences of different promoters need not be exactly the same, different promoter sequences typically do have some special chemical properties in common. Obviously, one property is that they are able to associate with an RNA polymerase. In addition, the promoter usually has a DNA sequence that facilitates the dissociation of the double stranded DNA such that the polymerase can begin reading and transcription the coding region. (Note: technically we could have broken down the properties of the promoter into design challenge subproblems. In this case we skipped it, but you should still be able to step backwards and create the problem statements and or relevant questions once you find out about promoters).

In nearly all cases, but particularly in eukaryotic systems the complex of proteins that assembles with the RNA polymerase at promoters (typically called the pre-initiation complex) can number in the tens of proteins. Each of these other proteins has specific function but this is far to too much detail to dive into for Bis2A.

A model of the E. coli pre-initiation complex. The sigma factor is colored red. The DNA is depicted as orange tubes and opposing ring structures. The rest of the pre-initiation complex is colored pink. Note that the DNA has regions of double helix and an open structure inside the PIC. Attribution: Structure derived from PDB coordinates (4YLN) Marc T. Facciotti (own work)

An abstract model of a generic transcriptional unit shown above with the addition of a promoter and PIC. Questions noted earlier that will likely not be covered in Bis2a have been grayed out. Facciotti (eie werk)

Suggested discussion

How does general transcription factor recognize a specific sequence of DNA? What might be the chemical basis for this? How, by contrast might the interaction of a protein or enzyme that interacts with DNA non-specifically differ? Think about functional groups and propose a hypothesis.

States of a regulated promoter

Since promoters recruit an RNA polymerase these sites and the assembly of the pre-initiation complex are obvious sites for regulating the first steps of gene expression. At the level of transcription initiation, we often classify promoter into one of three classes. The first is called constitutive. Constitutive promoters are generally not regulated very strongly. Their base state is "on". When the constitutive transcription from a promoter is very high (relative to most other promoters), we will colloquially call that promoter a "strong constitutive" promoter. By contrast, if the amount of transcription from a constitutive promoter is low (relative to most other promoters) we will call that promoter a "weak constitutive" promoter.

A second way to classify promoters by the use of the term activated or equivalently, induced. These interchangeable terms are used to describe promoters that are sensitive to some external stimulus and respond to said stimulus by increasing transcription. Activated promoters have a base state generally exhibits little to no transcription. Transcription is then "activated" in response to a stimulus - the stimulus turns the promoter "on".

Finally, the third term used to classify promoters is by the use of the term repressed. These promoters also respond to stimuli but do so by decreasing transcription. The base state for these promoters is some basal level of transcription and the stimulus acts to turn down or repress transcription. Transcription is "repressed" in response to a stimulus - the stimulus turns the promoter "off".

The examples given above assumed that a single stimulus acts to regulate promoters. While this is the simplest case, many promoters may integrate different types of information and may be alternately activated by some stimuli and repressed by other stimuli.

Transcription factors help to regulate the behavior of a promoter

How are promoters sensitive to external stimuli? Mechanistically, in both activation and repression, require regulatory proteins to change the transcriptional output of the gene being observed. The proteins responsible for helping to regulate expression are generally called transcription factors. The specific DNA sequences bound by transcription factors are often called operators and in many cases the operators are very close to the promoter sequences.

Here's where the nomenclature gets potentially confusing - particularly when comparing across bacterial and eukaryotic research. In

bacterial research

, if the transcription factor acts by binding DNA and the RNA polymerase in a way that increases transcription, then it is typically called an activator. If, by contrast, the transcription factor acts by binding DNA to repress or decrease transcription of the gene then it is called a repressor.

Why are the classifications of activator and repressor potentially problematic? These terms, describe in idealized single functions. While this may be true in the case of some transcription factors, in reality other transcription factors may act to activate gene expression in some conditions while repressing in other conditions. Some transcription factors will simply act to modulate expression either up or down depending on context rather than shutting transcription "off" or turning it completely "on". To circumvent some of this possible confusion, some of your instructors prefer to avoid using the terms activator and repressor and instead prefer to simply discuss the activity of transcription various transcription factors as either a positive or a negative influence on gene expression in specific cases. If these terms are used, you might hear your instructor saying that the transcription factor in question ACTS LIKE/AS a repressor or that it ACTS LIKE/AS an activator, taking care not to call it simply an activator or repressor. It is more likely however that you will hear them say that a transcription factor is acting to positively or negatively influence transcription.

CAUTION: Depending on your instructor, you may cover a few real biological examples of positive and negative regulatory mechanisms. These specific examples will use the common names of the transcription factors - since the the examples will typically be drawn from the bacterial literature, the names of the transcription factors may include the terms "repressor" or "activator". These names are relics of when they were first discovered. Try to spend more time examining the logic of how the system works than trying to commit to memory any special properties of that specific protein to all other cases with the same label. That is, just because a protein is labeled as a repressor does not mean that it exclusively acts as a negative regulator in all cases or that it interacts with external signals in the exact same was as the example.

Suggested discussion

What types of interactions do you think happen between the amino acids of the transcription factor and the double helix of the DNA? How do transcription factors recognize their binding site on the DNA?

Allosteric Modulators of Regulatory Proteins

The activity of many proteins, including regulatory proteins and various transcription factors, can be allosterically modulated various factors, including by the relative abundance of small molecules in the cell. These small molecules are often referred to as inducers of co-repressorsof co-activators and are often metabolites, such as lactose or tryptophan or small regulatory molecules, such as cAMP or GTP. These interactions allow the TF to be responsive to environmental conditions and to modulate its function accordingly. It is helping to make a decision about whether to transcribe a gene or not depending on the abundance of the environmental signal.

Let us imagine a negative transcriptional regulator. In the most simple case we've considered so far, transcription of gene with a binding site for this transcription factor would be low when the TF is present and high when the TF is absent. We can now add a small molecule to this model. In this case the small molecule is able to bind the negative transcriptional regulator through sets of complementary hydrogen and ionic bonds. In this first example we will consider the case where the binding of the small molecule to the TF induces a conformational change to the TF that severely reduces its ability to bind DNA. If this is the case, the negative regulator - once bound by its small molecule - would release from the DNA. This would thereby relieve the negative influence and lead to increased transcription. This regulatory logic might be appropriate to have evolved in the following scenario: a small molecule food-stuff is typically absent from the environment. Therefore, genes encoding enzymes that will degrade/use that food should be kept "off" most of the time to preserve the cellular energy that their synthesis would use. This could be accomplished by the action of a negative regulator. When the food-stuff appears in the environment it would be appropriate for the enzymes responsible for its processing to be expressed. The food-stuff could then act by binding to the negative regulator, changing the TF's conformation, causing its release from the DNA and turning on transcription of the processing enzymes.

An abstract model of a generic transcriptional unit regulated by a negative regulator whose activity is modulated by a small molecule (depicted by a star). In this case, binding of the small molecule causes the TF to release from the DNA. Facciotti (eie werk)

We can consider a second model for how a negatively acting TF might interact with a small molecule. In this case, the TF alone is unable to bind its regulatory site on the DNA. However, when a small molecule binds to the TF a conformational change occurs that reorients DNA binding amino-acids into the "correct" orientation for DNA binding. The TF-small molecule complex now binds to the DNA and acts to negatively influence transcription.

An abstract model of a generic transcriptional unit regulated by a negative regulator whose activity is modulated by a small molecule (depicted by a star). In this case, binding of the small molecule causes the TF to bind to the DNA. Facciotti (eie werk)

Note how the activity of the TF can be modulated in distinctly different ways by a small molecule. Depending on the protein, the binding of this external signal can either cause binding of the TF-small molecule complex to DNA OR binding of the small molecule can cause the release of the TF-small molecule complex from the DNA. The same types of examples can be worked up for a positive regulator.

In both cases proposed above, the binding of a small molecule to a TF will be dependent on how strongly the TF interacts with the small molecule. This will depend on the types and spatial orientation of the protein's chemical functional groups, their protonation states (if applicable), and the complementary functional groups on the small molecule. It should not be surprising, therefore, to learn that the binding of the small molecule to the TF will be dependent on various factors, including but not limited to the relative concentrations of small-molecule and TF and pH.

Is it positive or negative regulation?

Resolving a common point of confusion

At this point, it is not uncommon for many Bis2a students to be slightly confused about how to determine if a transcription factor is acting as a positive or negative regulator. This confusion often comes after a discussion of the possible modes that stimulus (i.e. small molecule) can influence the activity of a transcription factor. This is not too surprising. In the examples above, the binding of a effector molecule to a transcription factor could have one of two different effects: (1) binding of the effector molecule could induce a DNA-bound transcription factor to release from its binding site, derepressing a promoter, and "turning on" gene expression. (2) binding of the effector molecule to the transcription factor could induce the TF to bind to its DNA binding site, repressing transcription and "turning off" gene expression. In the first case it might appear that the TF is acting to positively regulate expression, while in the second example it might appear that the TF is acting negatively.

However, in both examples above, the TF is acting as a negative regulator. To determine this we look at what happens when the TF binds DNA (whether a small molecule is bound to the TF or not). In both cases, binding of the TF to DNA represses transcription. The TF is therefore acting as a negative regulator. A similar analysis can be done with positively acting TFs.

Note that in some cases a TF may act as a positive regulator at one promoter and negative regulator at a different promoter so describing the behavior of the TF on a per case basis is often important (reading too much from the name it has been assigned can be misleading sometimes). Other TF protein can act alternately as both positive or negative regulators of the same promoter depending on conditions. Again, describing the behavior of the TF specifically for each case is advised.

A genetic test for positive or negative regulatory function of a TF

How does one determine if a regulatory protein functions in a positive or negative way? A simple genetic test is to ask "what happens to expression if the regulatory protein is absent?" This can be accomplished by removing the coding gene for the transcription factor from the genome. If a transcription factor acts positively, then its presence is required to activate transcription. In its absence, there is no regulatory protein, therefore no activation, and the outcome is lower transcription levels of a target gene. The opposite is true for a transcription factor acting negatively. In its absence expression should be increased, because the gene keeping expression low is no longer around.

Termination of Transcription and RNA degradation

Termination of transcription in bacteria

Once a gene is transcribed, the bacterial polymerase needs to be instructed to dissociate from the DNA template and liberate the newly made mRNA. Depending on the gene being transcribed, there are two kinds of termination signals. One is protein-based and the other is RNA-based. Rho-dependent termination is controlled by the rho protein, which tracks along behind the polymerase on the growing mRNA chain. Near the end of the gene, the polymerase encounters a run of G nucleotides on the DNA template and it stalls. As a result, the rho protein collides with the polymerase. The interaction with rho releases the mRNA from the transcription bubble.

Rho-independent termination is controlled by specific sequences in the DNA template strand. As the polymerase nears the end of the gene being transcribed, it encounters a region rich in C–G nucleotides. The mRNA folds back on itself, and the complementary C–G nucleotides bind together. The result is a stable hairpin that causes the polymerase to stall as soon as it begins to transcribe a region rich in A–T nucleotides. The complementary U–A region of the mRNA transcript forms only a weak interaction with the template DNA. This, coupled with the stalled polymerase, induces enough instability for the core enzyme to break away and liberate the new mRNA transcript.

Termination of transcription in eukaryotes

The termination of transcription is different for the different polymerases. Unlike in prokaryotes, elongation by RNA polymerase II in eukaryotes takes place 1,000–2,000 nucleotides beyond the end of the gene being transcribed. This pre-mRNA tail is subsequently removed by cleavage during mRNA processing. On the other hand, RNA polymerases I and III require termination signals. Genes transcribed by RNA polymerase I contain a specific 18-nucleotide sequence that is recognized by a termination protein. The process of termination in RNA polymerase III involves an mRNA hairpin similar to rho-independent termination of transcription in prokaryotes.

Termination of transcription in archaea

Termination of transcription in the archaea is far less studied than in the other two domains of life and is still not well understood. While the functional details are likely to resemble mechanisms that have been seen in the other domains of life the details are beyond the scope of this course.

Degradation of RNA

The lifetimes of different RNA species in the cell can vary dramatically, from seconds to hours. The mean lifetime of mRNA can also vary dramatically depending on the organism. For instance, the median lifetime for mRNA in E coli is ~5 minutes. The half-life of mRNA in yeast is ~20 minutes and 600 minutes for human cells. Some of the degradation is "targeted". That is, some transcripts include a short sequence that targets them for RNA degrading enzymes, speeding the degradation rate. It doesn't take too much imagination to infer that this process might also be evolutionarily tuned for different genes. Simply realizing that degradation - and the tuning of degradation - can also be a factor in controlling the expression of a gene is sufficient for Bis2a.

Tuning termination

Like all other biological processes, the termination of transcription is not perfect. Sometimes, the RNA polymerase is able to read through terminator sequences and transcribe adjacent genes. This is particularly true in bacterial and archaeal genomes where the density of coding genes is high. This transcriptional read-through can have various effects but the most common two outcomes are: (1) If the adjacent genes are encoded on the same DNA strand as the actively transcribed gene, the adjacent genes may also be transcribed. (2) If the adjacent genes are transcribed on the opposite strand of the actively transcribed genes, RNA polymerase read-through by interfere with polymerases actively transcribing the neighboring gene. Not surprisingly, biology has in some instances evolved mechanisms that both act to minimize the influence of read-through and to take advantage of it. Therefore, the "strength" of a terminator - and its effectiveness of terminating transcription in a particular direction - are "tuned" by Nature and "used" (note the anthropomorphism in quotes) to regulate the expression of genes.

An abstract model of a full basic transcriptional unit and the various "parts" encoded on the DNA that may influence the expression of the gene. We expect students in Bis2A to be able to recreate a similar conceptual figure from memory. Facciotti (eie werk)

Summary of gene regulation

In the preceding text we have examined several ways to start solving some of the design challenges associated with regulating the amount of transcript that is created for a single coding region of the genome. We have looked in abstract terms at some of the processes responsible for controlling the initiation of transcription, how these may be made sensitive to environmental factors, and very briefly at the processes that terminate transcription and handle the active degradation of RNA. We have avoided more Each of these processes can be quantitatively tuned by nature to be "stronger" or "weaker". It is important to realize that the real values of "strength" (e.g. promoter strength, degradation rates, etc.) influence the behavior of the overall process in potentially functionally important ways.

Examples of Bacterial Gene Regulation

This section describes two examples of transcriptional regulation in bacteria. These are presented as illustrative examples. Use these examples to learn some basic principles about mechanisms of transcriptional regulation. Be on the lookout in class, in discussion, and in the study-guides for extensions of these ideas and use these to explain the regulatory mechanisms used for regulating other genes.

Gene Regulation Examples in E coli

The DNA of bacteria and archaea are usually organized into one or more circular chromosomes in the cytoplasm. The dense aggregate of DNA that can be seen in electron micrographs is called the nucleoid. In bacteria and archaea, genes, whose expression needs to be tightly coordinated (e.g. genes encoding proteins that are involved in the same biochemical pathway) are often grouped closely together in the genome. When the expression of multiple genes is controlled by the same promoter and a single transcript is produced these expression units are called operons. For example, in the bacterium Escherschia coli all of the genes needed to utilize lactose are encoded next to one another in the genome. This arrangement is called the lactose (or lac) operon. It is often the case in bacteria and archaea that nearly 50% of all genes are encoded into operons of two or more genes.

The Role of the Promoter

The first level of control of gene expression is at the promoter itself. Some promoters recruit RNA polymerase and turn those DNA-protein binding events into transcripts more efficiently than other promoters. This intrinsic property of a promoter, it's ability to produce transcript at a particular rate, is referred to as promoter strength. The stronger the promoter, the more RNA is made in any given time period. Promoter strength can be "tuned" by Nature in very small or very large steps by changing the nucleotide sequence the promoter (e.g. mutating the promoter). This results in families of promoters with different strengths that can be used to control the maximum rate of gene expression for certain genes.

UC Davis Undergraduate Connection:

A group of UC Davis students interested in synthetic biology used this idea to create synthetic promoter libraries for engineering microbes as part of their design project for the 2011 iGEM competition.

Example #1: Trp Operon

Logic for regulating tryptophan biosynthesis

E coli, like all organisms, needs to either synthesize or consume amino acids to survive. The amino acid tryptophan is one such amino acid. E. colican either import tryptophan from the environment (eating what it can scavenge from the world around it) or synthesize tryptophan de novo using enzymes that are encoded by five genes. These five genes are encoded next to each other in the E coli genome into what is called the tryptophan (trp) operon (Figure below). If tryptophan is present in the environment, then E coli does not need to synthesize it and the switch controlling the activation of the genes in the trp operon is switched off. However, when environmental tryptophan availability is low, the switch controlling the operon is turned on, transcription is initiated, the genes are expressed, and tryptophan is synthesized. See the figure and paragraphs below for a mechanistic explanation.

Organization of the trp operon

Five genomic regions encoding tryptophan biosynthesis enzymes are arranged sequentially on the chromosome and are under the control of a single promoter - they are organized into an operon. Just before the coding region is the transcriptional start site. This is, as the name implies, the location where the RNA polymerase starts a new transcript. The promoter sequence is further upstream of the transcriptional start site.

A DNA sequence called an "operator" is also encoded between the promoter and the first trp coding gene. This operator is the DNA sequence to which the transcription factor protein will bind.

A few more details regarding TF binding sites

It should be noted that the use of the term "operator" is limited to just a few regulatory systems and almost always refers to the binding site for a negatively acting transcription factor. Conceptually what you need to remember is that there are sites on the DNA that interact with regulatory proteins allowing them to perform their appropriate function (e.g. repress or activate transcription). This theme will be repeated universally across biology whether the "operator" term is used or not.

Moreover, while the specific examples you will be show depict TF binding sites in their known locations, these locations are not universal to all systems. Transcription factor binding sites can vary in location relative to the promoter. There are some patterns (e.g. positive regulators are often upstream of the promoter and negative regulators bind downstream), but these generalizations are not true for all cases. Again, the key thing to remember is that transcription factors (both positive and negatively acting) have binding sites with which they interact to help regulate the initiation of transcription by RNA polymerase.

The five genes that are needed to synthesize tryptophan in E. coli are located next to each other in the trp operon. When tryptophan is plentiful, two tryptophan molecules bind to the transcription factor and allow the TF-tryptophan complex to bind at the operator sequence. This physically blocks the RNA polymerase from transcribing the tryptophan biosynthesis genes. When tryptophan is absent, the transcription factor does not bind to the operator and the genes are transcribed.
Attribution: Marc T. Facciotti (own work)

Regulation of the trp operon

When tryptophan is present in the cell: two tryptophan molecules bind to the trp repressor protein. When tryptophan binds to the transcription factor it causes a conformational change in the protein which now allows the TF-tryptophan complex to bind to the trp operator sequence. Binding of the tryptophan–repressor complex at the operator physically prevents the RNA polymerase from binding, and transcribing the downstream genes. When tryptophan is not present in the cell, the transcription factor does not bind to the operator; therefore, the transcription proceeds, the tryptophan utilization genes are transcribed and translated, and tryptophan is thus synthesized.

Since the transcription factor actively binds to the operator to keep the genes turned off, the trp operon is said to be "negatively regulated" and the proteins that bind to the operator to silence trp expression are negative regulators.

Suggested discussion

Do you think that the constitutive expression levels of the trp operon are high or low? Hoekom?

Suggestion discussion

Suppose nature took a different approach to regulating the trp operon. Design a method for regulating the expression of the trp operon with a positive regulator instead of a negative regulator. (hint: we ask this kind of question all of the time on exams)

External link

Watch this video to learn more about the trp operon.

Example #2: The lac operon

Rationale for studying the lac operon

In this example, we examine the regulation of genes encoding proteins whose physiological role is to import and assimilate the disaccharide lactose, the lac operon. The story of the regulation of lac operon is a common example used in many introductory biology classes to illustrate basic principles of inducible gene regulation. We choose to describe this example second because it is, in our estimation, more complicated than the previous example involving the activity of a single negatively acting transcription factor. By contrast, the regulation of the lac operon is, in our opinion, a wonderful example of how the coordinated activity of both positive and negative regulators around the same promoter can be used to integrate multiple different sources of cellular information to regulate the expression of genes.

As you go through this example, keep in mind the last point. For many Bis2a instructors it is more important for you to learn the lac operon story and guiding principles than it is for you to memorize the logic table presented below. When this is the case, the instructor will usually make a point to let you know. These instructors often deliberately do NOT include exam questions about the lac operon. Rather they will test you on whether you understood the fundamental principles underlying the regulatory mechanisms that you study using the lac operon example. If it's not clear what the instructor wants you should ask.

The utilization of lactose

Lactose is a disaccharide composed of the hexoses glucose and galactose. It is commonly found in high abundance in milk and some milk products. As one can imagine, the disaccharide can be an important food-stuff for microbes that are able to utilize its two hexoses. coli is able to use multiple different sugars as energy and carbon sources, including lactose and the lac operon is a structure that encodes the genes necessary to acquire and process lactose from the local environment. Lactose, however, has not been frequently encountered by E coli during its evolution and therefore the genes of the lac operon must typically be repressed (i.e. "turned off") when lactose is absent. Driving transcription of these genes when lactose is absent would waste precious cellular energy. By contrast, when lactose is present, it would make logical sense for the genes responsible for the utilization of the sugar to be expressed (i.e. "turned on"). So far the story is very similar to that of the tryptophan operon described above.

However, there is a catch. Experiments conducted in the 1950's by Jacob and Monod clearly demonstrated that E coli prefers to utilize all the glucose present in the environment before it begins to utilize lactose. This means that the mechanism used to decide whether or not to express the lactose utilization genes must be able to integrate two types of information (1) the concentration of glucose and (2) the concentration of lactose. While this could theoretically be accomplished in multiple ways, we will examine how the lac operon accomplishes this by using multiple transcription factors.

The transcriptional regulators of the lac operon

Die lac repressor - a direct sensor of lactose

As noted, the lac operon normally has very low to no transcriptional output in the absence of lactose. This is due to two factors: (1) the constitutive promoter strength for the operon is relatively low and (2) the constant presence of the LacI repressor protein negatively influences transcription. This protein binds to the operator site near the promoter and blocks RNA polymerase from transcribing the lac operon genes. By contrast, if lactose is present, lactose will bind to the LacI protein, inducing a conformational change that prevents LacI-lactose complex from binding to its binding sites. Therefore, when lactose is present the negative regulatory LacI is not bound to the its binding site and transcription of lactose utilizing genes can proceed.

CAP protein - an indirect sensor of glucose

In E coli, when glucose levels drop, the small molecule cyclic AMP (cAMP) begins to accumulate in the cell. cAMP is a common signaling molecule that is involved in glucose and energy metabolism in many organisms. When glucose levels decline in the cell, the increasing concentrations of cAMP allow this compound to bind to the positive transcriptional regulator called catabolite activator protein (CAP) - also referred to as CRP. cAMP-CAP complex has many sites located throughout the E coli genome and many of these sites are located near the promoters of many operons that control the processing of various sugars.

In die lac operon, the cAMP-CAP binding site is located upstream of the promoter. Binding of cAMP-CAP to the DNA helps to recruit and retain RNA polymerase to the promoter. The increased occupancy of RNA polymerase to its promoter, in turn, results in increased transcriptional output. In this case the CAP protein is acting as a positive regulator.

Note that the CAP-cAMP complex can, in other operons, also act as a negative regulator depending upon where the binding site for CAP-cAMP complex is located relative to the RNA polymerase binding site.

Putting it all together: Inducing expression of the lac operon

For the lac operon to be activated, two conditions must be met. First, the level of glucose must be very low or non-existent. Second, lactose must be present. Only when glucose is absent and lactose is present will the lac operon be transcribed. When this condition is achieved the LacI-lactose complex dissociates the negative regulator from near the promoter, freeing the RNA polymerase to transcribe the operon's genes. Moreover, high cAMP (indirectly indicative of low glucose) levels trigger the formation of the CAP-cAMP complex. This TF-inducer pair now bind near the promoter and act to positively recruit the RNA polymerase. This added positive influence boosts transcriptional output and lactose can be efficiently utilized. The mechanistic output of other combinations of binary glucose and lactose conditions are descried in the table below and in the figure that follows.

Truth Table for Lac Operon

Transcription of the lac operon is carefully regulated so that its expression only occurs when glucose is limited and lactose is present to serve as an alternative fuel source.
Attribution: Marc T. Facciotti (own work)
Signals that Induce or Repress Transcription of the lac Operon
GlucoseCAP bindsLactoseRepressor bindsTranscription
+--+No
+-+-Some
-+-+No
-++-Yes

A more nuanced view of lac repressor function

The description of the lac repressor's function correctly describes the logic of the control mechanism used around the lac promoter. However, the molecular description of binding sites is a bit overly simplified. In reality the lac repressor has three similar, but not identical, binding sites called Operator 1, Operator 2, and Operator 3. Operator 1 is very close to the transcript start site (denoted +1). Operator 2 is located about +400nt into the coding region of the LacZ protein. Operator 3 is located about -80nt before the transcript start site (just "outside" of the CAP binding site).

The lac operon regulatory region depicting the promoter, three lac operators, and CAP binding site. The coding region for the Lac Z protein is also shown relative to the operator sequences. Note that two of the operators are in the protein coding region - there are multiple different types of information simultaneously encoded in the DNA.
Attribution: Marc T. Facciotti (own work)

The lac repressor tetramer (blue) depicted binding two operators on a strand of looped DNA (orange).
Attribution: Marc T. Facciotti (own work) - Adapted from Goodsell (https://pdb101.rcsb.org/motm/39)


Virology – Biology 3310/4310

This Columbia University virology course is offered each year in the spring semester.

Prerequisite: Two semesters of a rigorous, molecularly-oriented Introductory Biology course (such as C2005), or the Instructor’s permission ([email protected]).

Course Name: Virology
Sessions: M, W 4:10 – 5:25 PM
Start date: Wednesday, January 17, 2017
Points: 3
Location: Pupin 301
Course #: Biology UN3310.001 or GR4310.001
Instructor: Prof. V. Racaniello

Beskrywing

The basic thesis of the course is that all viruses adopt a common strategy. The strategy is simple:

1. Viral genomes are contained in metastable particles.

2. Genomes encode gene products that promote an infectious cycle (mechanisms for genomes to enter cells, replicate, and exit in particles).

3. Infection patterns range from benign to lethal infections can overcome or co-exist with host defenses.

Despite the apparent simplicity, the tactics evolved by particular virus families to survive and prosper are remarkable. This rich set of solutions to common problems in host/parasite interactions provides significant insight and powerful research tools. Virology has enabled a more detailed understanding of the structure and function of molecules, cells and organisms and has provided fundamental understanding of disease and virus evolution.

The course will emphasize the common reactions that must be completed by all viruses for successful reproduction within a host cell and survival and spread within a host population. The molecular basis of alternative reproductive cycles, the interactions of viruses with host organisms, and how these lead to disease are presented with examples drawn from a set of representative animal and human viruses, although selected bacterial viruses will be discussed.

Other course resources

1. Students should read Prof. Racaniello’s virology blog for information relevant to the course.

2. Students should listen to the weekly podcast “This Week in Virology”, produced by Prof. Racaniello, for additional material about viruses relevant to the course. You can subscribe to TWiV at iTunes.

3. Lecture slides (pdf) will be posted at this website before each class.

4. Videocasts of all lectures (slides plus audio) will be posted at this website.