of he greats lessons from the molecular biology er is th living cells use ‘commen st of design principles tht growin complexity bt retain basic xoms in progressing up the evolttnary te In this chapter the ccadian tur eon tues inside the pacemaker cls othe regulatory pacemaker genes and proteins. ‘The pathway to identity thee elements as een crt, eh many fale fads All sucesfl approaches have applied bse principles af eheonalogy to acti regulatory loops ‘Gti and malecular analyses have enti soa clock genes. Circadian stythns are based partly on negative feedback loops of processes. inherent oil ons the transcript and oe pots levels of specific lock genes la central role inthe generation ofthe hy. The proteins encoded by many ofthese loc nega tively feed back reduce the level oftheir own ransript, thereby providing an utotepultry system. Chapter 7 Hest reviews the log ofthe anal, hen exam ines the mechanism forthe best-known cases: the frit ly Drs, te fungus Nera, the yanabacterim Sysios, and he aborstory mouse MisMolecular Biology of Circadian Pacemaker Systems (One ring to rule then all... and inthe darkness bind them RR Token Introduction: Both Conserved and Divergent Elements Are Found Inthe Basie Clockworks"=" ‘Almost as soon a the phenomenon of ythmty was described, scents began theorizing about the basis the oxilations(Figu7), Early ead clock mee tls could not be very specie in term of molecular details because to litle was Iowa. The only ebsrvable deta ofthe lock wer is physiol and behoes ioral outputs ple he Hohavinr af the whole orilatony shim in eoponce te ete ‘al aeting cus Alf the four premelecularera mode shown in Figure? were ossbe Early theoretical analy sought to limi the spectrum of posible models 5a way of arying out cscrote bechemical experiments However saying that the ‘ieadlan clock probably represents a ead bop involving pie ar gave "ements not very revealing. The real questions concern fhe nature of fe he. ‘ments as wellas where an how they act Wire they within ces or berween cl? Daa the elements actin variety of ways in diferent Organisms? How can the com ponents ofthe lop be lentes? The dest approach depends onthe way the clock ‘mechanism is vislized. Consequently the experimen pathway tn en the mechanism oto clock have feted the views of any diferent scents. Despite divergence in approaches, however, the major question has ivy cor cerned the bcehereal, motculay and ella mechanism ofan ols. wit he ‘asc characteristics ofa creaian clock Especially challenging bave been the means of achieving the necessarily longtime const the means entrainment of the ‘oxilatr by vselight and varlous resting agents the mens of ateving tem erature compensation, and the nature of the acapement where temporal ior. ation generated bythe cxilator sed to ine he Behavior of he ell an rp ism. Within the past ve decades, anaers to moet hae questions have emeged atthe molec level214 Chapter 7 ~ ~ ane (wee [FIGURE TS fat aia pls ee etn lack models Fo farther An integral part of discussing what is known i review of wat Rabson done, ae why was cdone in {hat way Inthe fie of locks the descriptions of wht Und why give insight ito te resolution of one of the ajo es in chronology during several pst “Tee stermrang st te ulimate loved of resltion ‘Shether anytime are the product of itracellar oF Jecllle feedback loops. The material n previous ‘haters has already parialy resolved the tse By ‘Sob that the malstolar eye descbing a lee lal circadian esllator can be described within the hounds of sing cls without he nee to invoke ne alu communscaton between multiple cel The ute urourding hs question ecapsuated a mejor aspect of bow scents thought about rhythms Jint yours between 1960 se 1990, and therefore col ‘Sted how they approached the problem ofthe melecular fis ofthese ythos In 1960, when Erwin Binning fected the opening address atthe seminal Cold iringFacbor Symposium on Biological Clocks, the SEGRE Jocrton of he mecuae basi adn Si pened so daunting that he spoke merely ofthe SESE Rgecade tere foreword ta symposia vol dive stated "Ii ill very mach an open question ‘nthe not these salaries in pyscal lek chat reise common timekeeping mechaism at a | the alll evel i seems ily hat they ‘bret efit acommon mechs atthe fysiologicl snd biocheicalTevel in Flee organisms “Tosny tot the cock i a single-celled garam the Gono or Teale s ‘Tula as lway scemed olevidet. In ‘Sn amalmest np expof th ‘Casrended say thesame abouts mam alan cteadian cc, even afer the doa Rant oslo as oclzed othe SN, ‘he suprachinvnati nuceus. Moeover 8 ‘Tres view of the inteacollar versus ‘ntevcellla clock cones the choice of fystem stadia, the kinds of experiments “Tne and cen the expectation of acm tron mechanism for ythms ina vaney St phylogenetically diverse organisms Chapter outlined the bey experiments favoring an nacelle esas and a teitvely similar mechanism nal clocks. Remember however, that te commonal- {Yow seen sbaced on very salsa Fret speces stadt and ony avery ew cai aclatrcomponens have sc Giybenentie For Many Years Theory Dominated Experiment in the Generation of Models for the Feedback Loop Comprising the Clock Common characteristics of al oscilators provided intellectual focus to early work" ‘All osilatrs have thee asi equiremens: postive IMpurto dave change, fedback, anda delay inthe ‘tbcuson to feedback Regulatory mecianisms with these characteristics are prevalent everynere Facto [Tomb nes offen have feetsck contol rom end point to earlier point, and an aspect of industrial aaa is emineton of he resulting undesinble os {hoki ne ate of production of whatever is being ‘Ranuctred.Simulsey the metabolic pathways have Redback control exerted vis allosteric enzymes that ‘hange th chape ae therefore cr activity a5 sult ofthe ining of igands- I the yremotecr ers ren 1850 to 197 he prospect ofamigaie fneton win ipt eeback or delay to Sees Sholstoconcsly desrbe te micas Paty ein decane emt Ske 1950 hee SSS teal bctgy wad elecsinegnctie ve ‘ehetsted practically every aspect ofthe ie sciences, Ttuingdronetology. A sor Ustinches the esom- ant DNA fecnolope that allowed scientist fo the fim tone oan he gp between classical genetics at |Molecule Biology of Circadian Pacemaker Systems 215 biochemist in exe organisms. The new field of roecar gets every provided teaser ont how eels could bud ic operte a ciadianoultor with period length ofe day. Despite the application of molecular tools, the answers still depend on earlier eicadin insights, Including the concep! and universality of the pease sponse cv, the deny and pedo ction of ee. fing agents anc dhe uty of mice modal forthe ‘sls (e Chapter) The body of materia on ca Actes of ccadian oils xsi background forunderstanding thet molecular bass The pheolc pay it an example ofa imple feadieck crocs The well-known glyelytcoxilator is oughly enslo- {gous toa cradian pacemaker inte responses to per Tushations. The glyelyti pathovay provides carbon Hogment at metaboe ined for bieynthetic reetions and for the production of ATP The base be ‘hems is simple, censiting ofthe enzymes spon "befor turning pluore and 3 ADP into pyravae ad 2 ATP, while balandng the reducing tgulvalens (NAD/NADED in the res (igure 72) Although the system includes atest fv potenti orcas su ‘nding he allosteric mayen lyerldchyde+ phos hate dehydogenaes GAPDH) and phosphoracok. fase (PRK, PPK dominates flux trough fe pathway the renlting olan “The point af glycols iso make ATP and bisyn thetic intermediates. Te patra ergata aoe ingly by ATP and by crt, which is ey Intermed stein the citi acd ele into whic glycolysis feeds fatbon units In terme ofthe gycuyte oni the point ofthe fecdback ulation so make ATP when the concentration ito lo ano sop the production of ATP when too nach accurltes. [PEK rote ingyen to take «phosphate from [AtPand ad tt fuctse6 phosphate yielding Irae tose--diphosphatealus ADP Both product ofthe reaction activate PEK Consaqueny the more products Imakes the faster it worke™an example of positive feedback erating an elation In course of ach catalytic ejl, an ATP s usd and an ADP is produced The ection goes even ater and son the AP wed ‘up. When tis happey, he rat of production of ADP drops PPK sows down, and he eee complete. ‘Although th net product of glycolysis the ro- duction of2 ATP, thee are harvested actualy afew Step later in the patna, providing a lag The prod {ion of ADP and frac diphosp te alte both because ofthe inherent allostery ofthe enzyme and because ofthe ag provides by the mali stp the pthoeay. Actually sever things are oti nl Two ron 2ape Tes nam eet a {Su prt ofthese proces aaron plan ext Eee coeememets at irvabPonehaa pyetye celine cope shown wih back crows. 137A, L3-phesphosjcee SEED oelliowma So acces ramee ener Seis og epee cea {ng the acivityof PEK which changeson te ote of doa), the concentatons of ATP- ADE, fuctse-& ‘Phosphate, factse-L6 pe ae FIGURE70 Puting gone expreton nether. norman ese in the rales IDNA, whieh spiked i cst tively simple (igure 7.12, RNA polymerase can sg ‘zea na fo espe eguene fess found Ne Teglesing oa prokaryotic gees Th binding suf lent toate gene expression though the yess oF ons a : cay heneernmta? t ex @ FIGURE 71 The Cena Dog of moles lay. ‘apis of el oration ren outa by ance forte make RNA, wh then rane to make pote an RNA thats complementary tthe gene This RNA, Transcript icalled a messenger BNA or MRNA. Like DNA, RNA ismade by extension fom the end ofthe RNA/soa trnecripts made rom the ed the begin ing} t the en (the end), However, the binding of polymerase tothe promote soften not Sigh, withthe Fel that ile RN made Other ll poten aot { ransciponal cvatorsto help payers bind ot Ss raseripona epessrs to inhibit pelymerse fom Binding Asa fist approximation, subject a some ad ‘onal orstains and regulation, he more RNA thre, the more protein willbe made In prokaryotes asngle promoter can initiate tnascipen fan mRNA tat on Encode creo several proteins sil dese hee Transcription contines through the movement of te polymerase along the ONA vinta transcriptions fer ‘ination sequence ls encountered and the polymere bite the DNA hls Messenger RNA stranslted into protein in a very lange molecular machine called the rboeome, The #0 Subunits of the soeome bind the miRNA staring ne ‘hes endo the mRNA and move slong tas wn unt the fest AUG sequence tat an adenine felled by 2 ral followed by guanine—is encountered. This Sequence of bse ste signal o Dein poten syneFIGURE 72 Rast of poiycatvanic region conning wo genes ‘AG prokayee Th pms Ra pay mense ns to DNAte SubPStaranps wits nts con cont es protoing eine Suv by anime on ting the amin acid methionine al potas begin with ImetRionne, Ribosomes rean from the 5” end ofthe TIRNA, so tis end wl yea the beginning o the pro- fein Bocuse aming se ae polymerize through the ‘Gestion of namie nd between te COO” terinat- ing the extingproteinchain ac the NF ef the eco Ingamino aid esidue the stat of» pron the er ‘arising rr the potion of he RNA, an the fn of prin chains the C terminus aig fom a ‘hore 3 postion of them. From ere the sequence ‘of bases onthe MRNA ie decoded as sequential froups ‘he bases ae rend according othe genetic ode in which triplet codons designate specific amino acids (Figure 713). These aro ads ae linked in sequence Int the growing prota bound on the ribosome “The geneticcoe ie redundant in hat rote than ane distin ripe codon can ‘ouade the same amino ae buts pot Embiguous thai, each eiplet can be ‘decoded in only one way. Amino acids ‘te added by te bosome until STOP ‘oon (UAA, UAG, or UGA) isencoun- fovea, at whlch point the protein Ic released from the bosome. The ro oma However an remain bund tthe [RRNA and wlll move slang for while, fn fa second AUG is encountered the role proces of translation can begin enin The ego of tanecript between the sting AUG and the STOP codon ‘thatthe ribosome eds tn decodes sing the genetic decal am epen ead frame. serie of prove Coiing pions ust the conta of ingle promoter is Calle an operon and mRNAR that encode more than fe pro ae called polyistoni mRNA reins assume tee tee dimensional shape they ae sythesind in responses to rosa physical Foes and generally hey ae active once they have been ‘elessed lipo in ome cases atonal processing, (Guchas proteolysis) required to actvatea protein [Revit canbe fre nafed in prokaryotes wou postranslatonal covslent modifessons suchas phos- Phoryston of serine or threonine esidues Phosphory- Eon induces a negative charge (om the phosphate) ‘where formerly there was oly an uncharged hyo ‘group on the amino aid, an the result Etnies change nthe srt he pro- fer yeling incensed or decreased ate iy, qualitatively changed setivitis, oF lect ability to interact with other pro- {eine Phosphate groups aze added by Kinases and removed by phosphatases fn hey canbe add or emoved within minutes in response toa stimelas. Pro- tensa destroyed by proteases ht sever the pepide bondsbetween sine acids end re teletsing the individual amino acids for Inconporation ino other proteins or fur- ther metabolism ‘Several sent iflerences between eka vn ‘tesa prokaryotes ad to the compen eukaryote gee expression (BgU 7 19) Fite the fact that eukaryotic cls have Perrine) ‘Tyrosine | cytine gL vor = fc] ume | mate || cnr FE i = 4 a [| en men | om ES Ye ere [ee | ace FLL ase 2 se Te Bn Pl om ‘nore comple intemal suet, hing FIGURET19 ‘The gemiccode, lomston onan in pet coors in ENA bal eth langage pene spect ese sino Sor tosop nanan. eres of membrane-enclosed intra Srpanele betecen which proteins must tafe Second they general contin mach tore DNA than prokaryotes have Eecept‘Molecular Biology of Circadian Pacemaker Systems 227 2 2 Each primary transrpcontins on a ped ec ers ios) ot re ot ae vem sy tanta geoesincannt : tare sed eS © . {ode proine an hat ee a, Setar mate aa. a Posed pment ers) at malo up eae {re prod tamsrpt at en oon ing for rit Secure te removal fcchinon ia soperte set 1. Each primary tansript is xpped swith speci structe that wil ‘ll expor fom the nucle ad | sum STARE 510° urn Slherate ping may yl dierent _ingaganorm_j—*V 4 procened ranerp torn asingle Bene ech xpabe of icing the an Suess of diferent but partly, Spin cvelaping prong 5. Whereis in prokaryotes transcription alternator signa for polymerase {ofall te emplt,neukaryohee transrpaon continues inden, ‘bur theends of twansenpt are eter mmined by anther proesing event wherttvapaymensr tits Rite makes eoeape chev jets aterm dane eral wares roan ean be Talbert rtp ies Tre pala (AAAAS ta premonnl oftconsand ied he copack oe tens ‘ce smll amounts in the mitachonon and chorplst allot the DNA's contsned inthe ileus. Thus tfor mation wander beprsin theres, ain eukaryhes thencear proass of tanscripton spall sears ‘fom the toplsmie proceso tanelation Eukaryot have tee RNA polymerases, But only ‘one involved in the ysis of mRNA tbe ee fo ‘making prone Evkaryotes dot contain operons produce polyestonic mRNA stead each promater Srives ae musts expression of ust one pen cod Ing regio. Unlike the eae in prokaryotes, RNA poly erase isl eanno nite transcription but instead Feles on otter proteins in large comple, the bas traeciptionapparatis, to pepar "docking st” on 8 promoter thtalows to bind and t begin tanec on The astembly ofthis complen 2 major point of regulation o gene expression, Ths basal complex can recognize «promoter and bn in radientary way, Dut generally it does so poorly unles helped by sl ‘othe proeizs with high afi fr speci sles ia the Promoter rjin. Thee ar the dented transcription Factors that help assemble se complex withthe ply merase onthe prometer Asin prokaryotes thee ate tvators and repress, and diferent anscripton fac: toroen act together ona promoter ins combinational manner aft gee expres Once transcription as been inate, the primary transcript smade, and tiffs in several ways for those in prays Each ofthe folowing sep is su Fecttoincependent egulation In whi the primary ansctipt scat and sres of adenies re polymerized one te ¥ ‘nds of her wanserpt Th eukaryotic mRNAS "rn be dsingulshed rom other RNAsby the polyA tile Final processed trancrpts can contain ng ntalated regions before the AUG and alter the STOP coer, Proteins exist hat can bind these Untranslated regions (the and untranslated region) and confer rgulation onthe salty or Uuanslaton of the mRNAs Only fly processed anscrips ae exported for the nuceus ‘Once an mRNA hasbeen exported rom the nucle us, tan bound by thesome ed arlte.Th "ation of thi proesisubt to reyulason by proteins ‘hatean bind tthe ard untae gre Once translation begins the nascent prosin wl begin fo ‘sume shape, and bith this shape and the sequence ‘of amino acids in the fst pat ofthe pratt can cary ‘regulatory informationitecting the ribosome to halt Teaslation and go tothe endoparmic reticulum pot ‘oreuming synthesis: ote alae there re des ‘ned for insertion inte Bologial membranes or for ‘export fom the cell rnlaton continue fo be STOP fetion, the veslting protein can cary sequences of fino acids that es mansportof he pol ack nko the ncieus, rit various oer cellar compart. Because this informatio i contained in he ano acd sequence itis gently determined.228 Chapter 7 ‘Once translation has yen completed, proteins are sult to farther elation va coalent modification, fn the reper of ach moda slanger than n prokaryotes The means by which eukaryote proteins ‘ny be mode inca phosphoryl. methylation, nd acetylation al of which an change the local elec tes charge onthe sunfce ofthe poten and mol its ‘hope: atte ce neractions ith ther proteins y= Cosblation, the editions of sugars hat am influence \tecelr targeting and rego by her proteins: fn protoly either to destoy the protein or to mod ify tntoan active fonn, common covalent metic tom of protein in eukaryotes the alachment of Shot protein called ubigtin. Usiguitinaton marks 3 roe for anspor to and degradation in the pote Some huge presi complex whee faction it rap- idly degrade proteins Proteins are theultimate products of most genes, and changesin proteins ae the result of most PROTEINS AND PROTEIN DOMAINS. Proteins fold and sume shapes that refet thei functions and dictate theirncvies They arena rea way small molecular Irochnes, and they often sta they ha parts devo fc fo specie funetions. Such pars of proteins are hows as domains For istance,» protein hat acs a3 ranacriptional activator wil havea part (@ domain) howe leet recogize and speicaly bin on = Tain sequence of bases n DNA and another domain tnhowe fle to internet with RNA polymerase fo pro- Tote its activity. Often proteins work together in se ‘Se complees tocar out eatin asks in which case ‘Somainsof each protein inthe complexare designed to fever the pote poteininteracons that are equred tokeep the complex gett Tnpature often a variety of ferent domains cn exces the same futon fo instance proteins use at Teast half s dozen diferent domains to bind to DNA, ‘Stvealuntlated domains to interact with RNA poly- theron and variety of domaine to falliate pro- {Ein-protininteracons. One of the aera particular ‘Soman that promeits interactions between protein i= the DAS donain PAS domain are found i numerous proteins, Insome group they ae lo involve in sens Ii light oxygen, or voltage changes. Some FAS ‘domains also bind small molecules. rerstingy, PAS ‘Somain-conainng pron ae involved nll eukary- ‘fe etcadian locas wil be dese Ltr, ‘Domains assume thi structores a result ofthe sequence of amino sid in that pat ofthe prten For {henson the snquenee of sino ssidscan fen be usd to predict the activity of protein (oy virtue of Is Almminy) Because diferent domain quently canbe ‘Reed to peor the same funtion, the conservation of equencs and donmin tracts amen pti fm ‘ferent organism often taken as song evidence hat the protein evolved from a common ancestor and that {hey perform the some Fantions ibe se, Mutations ar heritable changes DNA; thats they tre permanent change inthe sequence of bases cathe ‘hrumowome In se eases mutations an besllen as forinstonce ifs sngle Bve change were to Ozu with than neon ats alvays removed om a spliced Wan Sapt nother iss ingle bse change can eu tmaprchange inte activity of protein Mutaionsean boss simple sa chang in one base toa ciferent one, fete addition or deletion ofa single base or multiple ‘ses. Mutations may aiobecompisted for xampe ehromasome sem may be broken of and attached ‘eihll of tr stendant genes, tothe end af a iferen ‘hromosome: Ina of hese cases, the change in the ‘Sequence of bases can rel ina change nthe chara tenets of the organism, Speciclly, everything ‘serie inthis short primer on gone cxpesio, from thestructte of infividal protein to the medics and roulation of ther expression, fe direct or indectly ‘Coed within the DNA. Aza esl al ofthese aspects ff potin sacs, atvty and mepulation ae subject “The effects of some mutations can be very subtle: A change fa sng bas can rsul inthe substitution of tne amano acd for another yeldng a protein hati Tite les sable st tevated temperate, othe mutant [Phenalype appears only when the organism expose Deteveted temperatures Alteratively, a ingle base ‘hang thatinteduced 9 STOP codon mediately ser the AUG sequense would probably resultin a complet Tos of fc ofthe protein Matton anise spon. taneously, oe aa rel of exposure to cherial mus fers phy mtagens ke UV Tight Regardless of the natre of the mutation, it sa change in the sequence of bases om 3 romesee Sit is amcited both with a phenotype and with «genetic trap oeaton and therefore the ONA containing ean etolaed and the nature ofthe atti described by eteminaton ofthe DNA sequence Understanding he ‘ature othe mutation ae Be resuing phenotype fen aks posi to obtain tformation about te phe romenon ing stain. For stance i ution that fave se toa Tong cadan period gt were ound 0 Five altered a phosphoryntion stein a prten, we Could ine: that phosphorylation ofthat protein ws Important in sone wa for determining peti length mporacly the ue of genetics and mutations to expo the nts involved sa process eed not assume Sything about the process. All that required in an ‘Srpanism isa phenotype to examine (ikea creadianMolecular Biology of Circadian Pacemaker Systems 229 ‘yth) ands, the ably tosegreate genes in gent: Sceroses and thst map the If tes spl ee ‘which gene are important forthe phenome, ‘An Understanding of the Molecular Bases for Circadian Rhythmicity is Emerging’ ‘Common themes amang chcadlan oscilatos are cevident=™" [NEGATIVE FEEDBACK LOOPS. One common propety of tn osellation nt tendency in a regular manner to ‘ove aay fom equ fore returng Negative feedback sone wa to atee this temporary dept ‘ure fom equlibium, All tats needed isa process wise product feeds back to slow down the ate a the process itself @ negative element), and a delay inthe tecuton of the feck Big olatrs could be ble rom varius itferent regulstory schemes, a= reflected isthe wide array of imaginative models ascribed eae hat were proposed inthe pre molec: ‘argent eras bse forced yt. A met boc pathway ov anion flux could in theory work at ‘well ae ranecipton and/or anslton ko achive ne ive feedback: Delays could result fom hysteresis» Slonnes of response yieldingen overshoot when equ [via is being approached or fom a threshold pe nomenon preventing immedi feedback, tna ly tion osilator sucha ppete washer. Yet snother means of achieving delays through nonlinearity in "Reponses os when mulliple exponen mst nd each ther and associate prio 0 eectng feedback Abi logis! osiatr must alsa hive s positive element, sure of excitation or actvaton tat hes the oxi {oreo winding down ally ntheory ofcourse feedback ops that ope ste twcen cls ue neuronal conection shold work Just as well as intracellular loops for bung 3 lock Sch intercellular ils re ery corm ine re lation of hythmic behaviors such as wing oe an Intrigsingly however alclacks have wolves wouse ‘ery simular molecule solitons to the feedback ad ‘ely problem All own can esiltors se fas Ick oops tat le within ells name req cell-cell Interactions), and all lyon positive an negative le ‘ent in oellators. ceca clock loops the tan ‘erpion of clock genes actated by postive elements tne yields clock proteins thatct as negative elements ‘These in tur block the actcn of postive element) whe role sto activate the cick genes) gute 713). CGENERALZATIONS. fn no onganiam do we understand enough about the general sembly of radian sy tem tobe able io seriously contemplate amin vito oF even an in vivo reconstruction of circadian clock, However, workto date doco some generalizations feedback loop that inva gees whose products te not eset fife Insts the function of thee genes devoted solely to cncaian timekeeping in keeping withthe evidence that lacks are adaptive but not ex {alto ee Chapter 2) Second many ccadian sje ‘ems will probably involve the interaction of multiple feedback loops although at present only afew ofthese ‘an be decribed in concrete tern. Tis the Cocks ny feganisms among the eukaryotes that share a most ‘ecent common ancestor wil abate lente and sin iar components inthe arsembly and operation of ther locks, The groups ae inorder of lt, the an ‘mals fang and probably higher pants "The expacty for cicadian timekeeping probably arose more than once evolution ut tere Sgest that i arose nly a few tines. Within the eukaryotes studied» stn steam of functional and Sequence based sires have emerged as the mc Ua bases of ceadianeeiltory systems have become Understood (ee Figure 715) Ths delibertly overs plifed moda an serve as reference pint for unde. ann the overall onganzation os eae lop tha foe part ofl known lacks, A generic core circadian feedback loop can be desenbea™ ‘The schematic view of core oscillator model that is presented in Figure 18 shows the common elements of the fedack loops that have been described 0 fy ‘which are generally taken a ne of Ue core calatory loops of eteaian sytem One might intrpet Figure 78s implying tna radian ceca wil be aly simple wanscription and translation eck Lops, but they wil probably not be ths imple "The potve element i th lop i the anscrption alactvaton of ene or more dock genes In he cyano: tertum Syrecioacee, one model predicts tat lock iene wil be activate bythe KatA gene product(s er see the diraion that lls for abe interpret Hons) In eukaryotes such a animale ana fang, clock bones ae apparently activated trough the binding of (Reisen FIGURE 715 Thebaicelements in ceadan edback230 Chapter 7 transcriptional activators which re pred via interac ‘None of PAS dorsi within he protein These poitve ‘ements bind fo DNA on te cock gne promoters 0 Sctvate clack gone tansctpton, Eunsonaly analogous PAS domain-onlaining DNA-binding lock elements trith similar overall protein sequences have Been ‘ose inthe tive ean onan —Newaje, Drie, and muse whose clos have ben sted the mos thoroughly atte olacular evel llth will tedesrbed ere nde These petive clan drive Aanscription of clock genes tha give rise to MRNAS ‘whose transation geneaes ack proteins that prove the negative element inthe feedPack loop. Examples Ince the Kai gene pnt n Syrians FR in [Nearsjor PER an TIMin fs and spore PER PERG, CRY and CRY2 mara "The nepitve lementn the lop feeds back to block ‘sctvation of the clock genes, ith he tel hat the Amount of dock gane mRNA, and eventually the amount ‘flock protein deine As the oop cles generates ‘ell inhibition of rarcrption factors (the postive ele ‘ents The action of hese postive elements on other ‘ock-controled genes sugget that tie tortion fromthe calla ight deve ouput by regulating tar get dock-contlled genes, as willbe decribed more {Ghapr Ths robust daly eying dock ge mRNA, ‘Sock protein, an lokontolit gene RNA and pro tein characteristic of cada systems Evidence up poring this loop 8 cor of creation ceils is theineral consistency fhe ude genic: Most {genes that have bean identified in cyancbactr, Nevrasora, Drape and plats bs genes that ‘Mec crcadian cocks ca bet ins this framework “Th central importasce of thas loops also sup ported by the fact that environmental fects on these Eomponents hve been shosen tinder resetting of the ck cyte by ight and temperature Although ot all ofthe detail of all systema fom cjanobactera ‘hough ang though humans ave yetbeen decribed, ‘many ofthe elements meioned here are known i all ofthe systems that have sen examined. The tveads of Smarty among al systems stongly suggest that ths feedback oop reflects scommon mechanistic cor for ‘most ifnotall neages ef readin oscil ‘The Cyanobacterial Clock Depends on a Negative Feedback Loop Involving ‘Transcription, Transiation, and Protein-Protein Interactions [Neal eveything korn about the molec basis of {yanobscteral locks ermes rom sts on Sythe ‘ess lait neyanebactera, Be loc can elke {'vatey of proceses, eluding cell division, amino ROURE 736 Calo of he cyberia pcre Syne ek roasted poate lies eee ‘Sons gringo pet ie fe dt a te Sica ie ere ehh pa Te se ‘end dag he nee pte wes viewed and cad ‘rid yp cle be our dint sens ra ‘Rowe Mode om an srg ihaeatanty T Renda) ‘cil uptake, nitrogen sation, photosynthesis cabo Inytate synthesis and respon The particular uty ‘ofa ming spe i manera inthe nt wo of es In which the itrogenze enayne required for mitogen fuation is poisoned by the enygen evolved from pho symesis in fact both process ae vedio ep [aed 180 degrees out of phase (se Chapter 8) Mutations in genes that affect operation ofthe Synenccs clack were blared na Bouminesence reporter screen in which clockregulated photos {etic gene promoter (js) wae fuse fo bates lateral used to dive shyhni luminescence (Figure 716) More than 50 tants ware dente, ‘ving period lengths ranging from 14 to 6 These ‘genes hve ben cloned and shun to compris cle {St tvee genes knw asthe gene the Japanese ‘word for "cycle" (Figure 7.17). Expression of the [Benes i driven from two promoters, one Fr kia on ‘ne for both 8 and The functions of heh gees can be inferred from their regulalion and from the phenotypes of alee, Deleon or overexpression of te or fBC rele In athythncy Dut atin the ste wy. The Kal pro- teins constitutively expressed and loss function Iiations of lait resaltin very low-level arrhythmic "xpresson from the ini promoter Overexpression of [nu yields constant superleates expression of BC sd, gai, aetythmiy. These data Set at KA 05 ab an activator af teanserpion. KaiB ad CareMolecular Biology of Circadian Pacemaker Systems 231 We eS Sd artista wasted and eran ecko {Srabie wth other prtns oom complexes which my case soba changes ep peng oncoming he bc cena Tn Ere feedback on. Pay end Pg tert the promoter oh ere oh sumably work with Sash has not Yetlsen ented, this oto ts Well with the known eects of iC on raneponal ln, [SiC operon (erie and non S01) ‘ythmiclly expressed with» peak at about cradian time (CT) 18, and te lines ofevidence speak to thei fnetion, Fist inactivation of ether abot i andi yes archytuncty and altered expression fom the [ni8c promoter Second, overexpression of iC ele Inantiyhmty andi severely dampened expression of ini8C but not of EA Ti, pulse producion of iC rests the cecilaon. Thos dat a consistent wih {rob forthe KalC gene product ars negative elaent the ltr: no see can current be asigned 19 Kal All fhe Kal proteins interac, nel tations that alter thes interactions afc the operation fhe loc, Forindance, te interaction between Kia and Ka is Strengihened by KaiG. Also the strength of the KalB-KaiC interaction oils with the tm of day, and 2 mutation tat att strengthen the Kaka Imteraction lengthens te period of the ack ‘Screns for mutation in genes eneong other pro ‘ein tht interac with the Kat proteins have reveled ditional cock elements, among them SaeA lor Synchscs adaptive sensor") Sanit hse ro tein Kinase fhe type ually associated witha DNA Dining earserpion mgslting response repr” a bacterial wrcomponent signaling pa such to femponent systems ne component senses a ean ‘mental change and becomes seated it then phospho ‘ates the second component tiger a esponce. Loss OSs does no black thie but doer elt tenfold reaction in fC RNA olations asf Sas ‘worked san amplifier, Transient overexpsion of Ss Wil phase-sht the cock suggesting t cental role DAivnough the cognate response regulator tht would poe ‘The KAC potainhae an ATD/GTP rucleotide-inding sctvty, and ai phosphorylate ap part ot actvy in the lock, ‘One ofthe mest intresting aspects the yanabac- terial lock that teal in shytmicransaipton of all genes inthe cell, suggesting global regulatory ‘mechanism (soe Chapter) Siilartiey between the Btnin aed sequence of KaiC anil bacterial helices has Suggested the pouty that Kei could at sah fase directly onthe chromoromal DNA to change the Alegice of condensation. Helcaes in general st by dingo removing twists ia the DNA dou hl in genera the more ihly tse the DNA the ote dificult for ONAsdependent RNA polymerase © leanserbe the genes Thin his model Kai mea. ec cicedian regulation of the degree of DNA cling ould globally influence gene expesion ‘Genetic screens are alno beginning to suggest le iments of height input pathway though which the ‘yancbaceral lacks eet Among possible input dle ‘ents is CikA (for “cradlan input nage") protein That bears sequence similarity tobacerophyochvomes and histidine kinases, Los of CIkA shortens period fects phases of some cyte and alle entanent to dark pulses by reducing the phase reponse curve fo ‘one-fourth ts normal ampite, a well as making it ‘spas eth the ld ype. Considering that a decade ago nothing was known about the molecular bass of eyansbacel yon Is ea that gest progress has eon mae, ever hough much remain tobe done. Many ofthe ompenents in the feedback lop are stil tbe denied a he go ‘ering principle ft oop yet oe imine Coe repolatory themes, however include ceadin ep232 Chapter 7 ton of tansrption and ranciation and the importance ot phosphoryation and protein-protein interactions, ‘which may influence each oes, Generic Core Circadian Feedback Loops in Eukaryotes involve Interlocking Positive and Negative Regulation of Gene Expression” ‘The fis icadian feedback loops tobe understood ‘moleculssly were those in the eukaryotic model sy {Ems Newrogpoe and Drosha The systems share Some similarities and ferences with each the aed tritheyanobacter, but they serve well introduce he ditional compesty of eukaryotic locks. Asin the (jancbacteria,mythmic expression ofthe transept Shu proteins comesponding to the negative elements is the ule All known ieadianosllators involve nega "ve nealtion of ranseripson asa prof the loop ut Inthe syanobacteral lock it was not lear how that regatve regulation ws proded. In eukaryotes his ‘epative feedbacks known to occur through action of the negative elements in blocking the transcriptional ttivatingsblity ofthe positive clement (Figure 718). AA postive elements are heterodimers composed of feo lferentprotens that ntoract via PAS domains (ne ofthese to proteins is constitutively expressed, Shu he other rytumialy expressed. In adtion to ‘ornmon Function snd regulation, extended sinlaity [Senenin fhe amino ai seqenens of some the po itive cement proteins Finally an all non cases, one of he nopaive elements as a dull nthe cyclen that performs both the negative function of bocking Fhenely af the trader and alo a positive fun ee FIGURE 7.8 Features in common among te moteclaly ttespentsnton neon ratn cbr epeson ofthe negatve mer prin at toto inact a epost let we in Fromatng th xpmion um Dun 198) tion in promoting the expression of one ofthe Ft ‘rome partnea: This dilaton dg the ne tive and poate limbs ofthe jee ceetesinertoekng regulatory loops, ne scen when FRQ promotes the xpression of WC, PER/TIM the expression of CEC, RIPPER? tne expresion of BMALI. Ceadin ouput {ate Chapter 8) Gerves in part from the action ofthe {lock regulated pole elements onthe expression of {genes whore products do nt fed back ont the etal oc lop ‘ABBasic Eukaryotic Clock in Neurospora Uses Several Components to Execute Negative and Positive Feedback" ‘Both Neurospora and Droop are model systems in twice tls nd paradigms necessary forthe molec Uhnrdsscion af erendin ing systems wee devel- ‘ope. Newrosjore normally grows as a syncytia in hich many nucle! share a common cytoplasm in @ Typha, an individual lament (ee Plate 2: Phat), When cultures sre gsown on 9 rlid substrate, the [Newospor lock conto the pattern af asexual deve pment in the segon of the growing front, Arial ype, which in devel ino vegetative spores led Conia are fom mye aid down in the ate rght through erly momting In eontast yeti id down othr times of day do ot generally develop further bt intd emain nrg need Figur 119) “Athough many molecular outpts ar known and bo Tominescence bese porters have been developed for monitoring shyt recy ml eltures this Thythmi change n growth Rat emai the most v= ‘ousmanietation of the Neva clock. The credian ‘ature of tis developmental site fom suc ae ‘Bigowth and dferentstion was noted by Pitentigh ‘over ynts gos was used by Feldman in theealy 970s to dentfy the Best Newompoa lock-mutant tain alles of the fue J) gene {Essential components of a circadian oscilatr, feedback loop in Neurospora are known’ ‘oveRWE, The Neue ccadanosilstor includes {troche postive and negative fedbsck loop used ‘Sr transcipton and talon witha heterodimer of PAS domate-conaiing protein acing a arrp- tonal ntvator and a single gene encoding negative ‘ements, Among the element hat consti the o> Iatorae the WCC (white coll") and WC-2 (white Collar) proteins, as wel a both jy mRNA ar the RQ protein, and soverl kinases (Figure 7.20. WC nd WC heterodimeric to form the whe cll oo flow or WCC The intestions between these Wo df= Keren protein are mediated by PAS domains in achMolecular ology of Circadian Pacemaker Systems 238 lubes, Stains arent on tet gro or yin thei andre carne ih epee se Sysco proce nec pecans ‘cral hype are no lier produced. The growth ron thas Sedbothensal dete rw SE ceo see [cin The WCC activates expression ofthe fy gene. The fo mRNA then encode to dtnt FRG pros (ha fed back to block sachin. Las of function rtations inf, 7, we? esl ‘nos of normal endian fy, though son lcadan elation emain nthe backgroud ot isthe rps. This mca that dona ed bck loops exit, but hether they are involved inci casian tytn bal unkown, Mutations nfo 2ikat preserve sulient function to stan cadian ‘hythmisty can rel insubstantial period Tength defects, yielding periods from 16 1038 8 wells ‘ouling in paral er of temperature and ating! compensation of the ick Both ey mRNA aed FRQ protinarehythmialy expres ina dal fashion, {ind FRG repens te apundance fis own anscrp. Inpho on eles Tis cy curs evry 21.6h and ‘sly ot tasters mek ogee emma see esa gh race coed ly ining bande Feng a fe cck simpy ‘ee ene {tates nthe pening fan rea eo ‘hoje gent i hci ae al Rie tor ne ge CCENERATION OF THE CIRCADIAN OSCILLATION. With Figure 7202s guide, the progzess othe Neurospora lock jee can be racked fom tea ight At ths Ge most of the FRO protein in the cell has recat been Alegre and fy RNA levee ae low but ae begining, torte. A period of 10s required for hg mRNA level to rise fom thelr ough to peak amounts, The tan Sexpion factors WCL and WE2, athe WC. bind to sites (Clock Bowes) within the prometer ofthe fy gene to dive the ciecion shyt in tasctption of fy. AS the ig eanserpt is produced, the inal primary wan scp aresplce na temperntre dependent ene Gradually by early morning, FRO proteins eppear ‘thera fang ora sot for of FRO canbe translated, depending on the prsucts of the empertire ela ‘ediaterative splicing of th fy waneoyna eer cpanel er See ee oe mecca mere te nee Tene ate onemeet te Sage panier clereaar et Sree aries ace tare SRS eg aReESL Seaton Seer et eee it Ca Fearne A een et EES ee Se ee The FRO proteins made son form both homosdiers ne btrodier and they pareipate in thre separate “Scion tha ogeter govern aspects ofthe yee Fist RQ eres the maces wherein inspec ways tnith the WC Because tbe WCC-DNA complex on he Jrapromoter does not contain FRO, itis ike that FRO ‘urs its negative le inthe clock cycle by seducing or pecveting he sity of he WC tobing tote Coe Bones inthe fry promoter By midday WCC activity is ecg ots awst evel jst as FR levels ce ising tape te to tum down the expresion the re ge ‘Second, nd independent FRQ begins tebe phos phhoryatd by several Knaes inelding casein Kiaes {Mid 2 soon asi made This phosphorylation is Tesportant for determining the stat ofthe FRO pro- tei hich in tir determines the petiod length ofthe ‘SocieAt his pin close wo midday, RQ levels ace igh ‘and continuing tse; FRO s becoming phosphoylat- ‘Shand has enered the nucle to bind othe WEE. In Swld-ype cll at oom temperstre 23°C), typically ‘out 4 molecules of FRO wl exis wethin He mucus fren FRQ levels are a thee peak, compared a the he time tos quarter that many molecules of WC: Sd more than tre timer that many of WC:2. As 2 ‘esl of FRG binding the WCC aa reuing its at 0 by we mae anion of fig (CT) Level NA pak Sail Phgoppanc tegen mae te in ine mr ge un and phosphor [oy fons FO pres he sre WC whe Feeminga inane erpin wih he WO hpi a {ie (CY ta) Conte php aes FEO isla type egrdes oa he ‘WE io cute ry eprnton sds tec ane ‘Eekconled gone fem Delp 198), iy fo expression falls fy rans levels begin to ‘ecline, Ongoing trenton of the remaining fy InRNA, however slows FQ prot levels to cone {ose for several Hours so Dati mRNA evel peak in the midmoming, about to 6 hbefore he peak ota FEQ in theafteroon “A about ht th third oe of FRO in the eee semis FR promous tough an unknown me ris, the lanalaton of WC from exsting el [SIRNA Fortis resson levels of WC-1 bein tose ‘ren phosphorylation promoted degradation of FEQ [ogine. Tuwat close to he same me FRQ Blocking fctivation ofthe fr premter by interacting with he WCC whe promoting HC-1 sythess to ierease the level ofthe WCC. FRQ sso promotes the syiesis of WC2, although perhaps sa result its stability the levels of WE do note, Because WC-2ip aaa scone high tls, the enfiancement of WC ‘Synthess by FRO cess an ever increasing amount of WCC, but tis WCC Ise eld inactive when FRO. ‘duces the ably ofthe WCC to bind DNA nally the phoephoylson of FRO ulimately tg erst precipitous turer and the WCC is eleased {WA levee pein the night nea to when FRO eves opto ter point Tis wave of WC-1 cated by theMolecular Biology of CicadianPacemaker Systems 235 Junaposion of FRO promated WC: syn- (A Retr wth eh these and the blockage ef WCC activation a tah creatsa sharp ansiton, With ighWCC —Youvindwk 28 32 Seto ae se ae a scion the ext cyano fain mbat ample nthe feed oop nos Trsummary the Netra clock on 3 tains postive and negative esac oops that te interiocked 3 the mull ach tis oF FRO in promating WC-1 synthesis ile blocking WCC activity. The het Eeawccmsnsems AQ wwe negative iment The molecular basis of entrainment to light and temperature changes is Understood in Neurospora" The go stn to move dy Paseleck acne ool Percasar cate “hn a eek Inthe eu yest Re anue he meta ot ‘earn ighitatheane es Cen osha pont hb Se eee the iftscponse apse an tpt meted ue Chape SEM Nn sr ‘igh ectp ind pvp cong Win cmpenns tpn e anmccamereemnn }\ = hauled nhc pe Srrapiy tough thewCein SCUNET2, Reig Nevo ce, peg oe er Intact ncrcipgenrencn Sahih Rag ante Neha Seale tp ty Set eeoe argieay tc gu Sea ee ‘ee WEtidetAD (heey is dear arroyo toe) Piney cmaianiowepe: | een eed ec saree oy Re FAp cn tele, Eric comtearig eemiene nae arate sagen sconee mwcandin hale es cena ne epthe wasp tf ore eflectvely Bee ight sede ecto peakpc en Sells pried cet nabrybocieey tretinoin gh enya ey Sarsimoming fake econo big Ispukterch qty sana he cs buat teperlngtsuotng Comeech tea es FRlveevening un ei ight whe yma es ‘efting ncn tsp post sndends then theese eos peak comapeding to niey linge pe dey Getic Zn) Te haa ighceaprepese ato gated tcl soa oe ines Sy Sina hein me conf ok Stereo ser resin! eye {ik pin VO) wb epi ms Supe “Kb temper nes hymn ey czl ayes Repo: in Sep pin ere trite cink none sea igh pes Sondre physloa mpesie oe spermine he Love nga ete ofersin ‘Sp ed nh ne ees eg fovea he me Ths whe cuaecork peers Town as tempetae compensation. Whe light tarsrptona pullen ty terre ‘es ar medi tag te aout chaps thereat FRO pin nic emer tito tal amount ARQ el cee {of thelong vem sort ne Wen be Wy of heel emake eer 2 arn ed Sash maton te amp ane perme ar ‘yoni rte Ta pone aspect iny seve oxen the pyslopeal tonne tage oe hh he deck cone esting te ok epee sips als st otal mae ere Perea san bat RG amounts oan ghar Nigerenpennues Fgue 2B hows for tour 228 th opercett Ngu 7210) loves pot inthey jen 0 abe igi thn hes pontine crete apo 2 thetower cure igs 73) Bes tee FROin ocular events tes the’ ean ie sce! ws gen ese tokesiof PRO dec a dest ones GeeFigue’20) Tain 35 maou thence peak sun seve soho rornioacinincins tems incase nate anaes hoeve se tp ee FRG Iore oly ccc of RQ ences igh ‘Site pant ee seep This scm sin tenpersta cares o asta nat the cok aly sscpee ae ferent time) although ntly no synthesis or tumover ‘of compononts occur. After the tenipertare step fl. five levels off mRNA and FRO ae selene y he ‘itcadian regulatory cuty in ets othe new fem Perture and they respond apa If there oo ie RQ to cause negative feedback then it must be sje live daytime and more fs madi thet le more tan ‘enough FRQ to block ig mRNA expresso then fmt be nighttime and nane is made unt some FRG hes Segraded “Thus, unlike light, which acts via a photoreceptor cutie the loop, temperature changes atthe Sad ‘cycle instantaneously and rom within, withthe ro Ulatory dynamics ofthe cycle el acing a the toa erate sensor In the natural world, temperate ‘hanges seen at dusk and den can approximate step ‘anges, nd surpiigl such nonexten temperate ‘anges in Nexrspom and 3 variety of ther organ Sem can havea stoner iflunce onc tiring, ‘han ght (ee Chapter). Inalleass though, ligand temperature cues reinforce eachother to keep clocks synchronous inthe al worl ‘The Clockwork in Drosophila, a Model ‘Animal, Relies on Patterns of Gene Regulation and Feedback Similar to ‘Neurospora but Reflects Increased Complexity:”5012 ke Neursporn, Drs paradigmatic molecule ‘iwcadian system whose development has ilminated the tall ofthe operation of clack atthe molecule level. As outlined in Chapter 3, early work by Pit tendrigh on Deselect desestptons ofthe formalisms il wed for desing yrs The thythmic characteristic that drove ery escerch wos pupal elsion emagence or "halching) which aes Place ina tightly defined window of tne ne subjee- five dawn (gure7.22) butn recent decades sts on Disa melngaster ave flowed he daly te- psc (dan and dusk) ay in locomotor act ‘Genetic analysis of rhythms in Droop began ‘with the work of Konopk inthe erly 1990s: po eat the fist cock gen he denied, and the ist one tobe ‘ned a decade ate: Much of wha s known about ‘hythas in general desves rom the study ofthis gone ‘regulation and its products Frit fle ave levels of complet assoated ih tatu speilzaton of ine tion that have offered insights into the ccedien sys. tems of multicellular animals beyond tose avaale ftom Neirospor or other microbial system, demon stating, for instance, thet ell utonotous cocks at found in most sepacate parts ofthe adult fy (ae Chapter}Moleculr Biology of Circadian Pacemaker Systems 237 FAGURE?.22 Rhythmic behavior wi elapse, (A) Hatangircoe cmos and sorely sot ce ae ‘Stmanp eggs hah dar tnesagpctive day ped me sgeine ‘Many central components ofthe Drosophila Circadian oscillator are known "0" COvEnvEw. The Drop ciccadian oscillator includes Inicocke postive and negative feback ops based tn earsripton and tration, witha heterodimer of PAS domair-contining protein acting arse tional activator and two genes encoding negative ee ‘mont in the fedback loop The cove intsacked fed Back loops are defined by the ations af the CLOCK (CLG ane CYCLE (CVO) proteins, prin pr) and tine tnd ter movers ska sam igh Te leg wih computers (Afro Kosoprand Bena, 1971) es (in) mRNAs, and PER and TIM proteins Ths are aida by the acon of VRILLE(VRI) an thre Kina ‘CASEIN KINASE 2(CK2), DOUBLE-TIME (D8), and SHAGGY (SCG) (Figure 723). CLK. and. CVC het ‘erodimerze to forma comp (ln Figure 7.23, the (CUK-CVC compen or CCC} the anda PAS-FAS et ‘odie feuuryetic cena lock loops that aa ‘gous fo the WCC in Neapore, CC activates expe ‘Seva the pra tin genes, which then ence potas that sult nfeetback fo lack her own atti. PER appessto bethe potas actualy crs pes Is iacelllr oeaton. Loswo-lnction mutations (Che eo tin el of nal cain yt ‘mii although noncireadian osllations have been ‘ported inthe background of nll strains, DBT ‘quired to phosphorylate PER, leding tis degrad. ‘on, and phosphorylation of TM by SCC promotes nucle entry of TIME PER VR bins te Cir promot to depres Cle expression and thereby to repress e od tn RNA scumulton,CK2 probably acs opine phosphorylation of PER and TIA by DBT and SCC Pal ossoPfascion and pin ofncon mutations in238 Chapter 7 le we = commpnaaa | scar sop ci Sec et ee re atin afeeme tsa, eer er Seiaile creamer any ofthese genes can result insubstantial defects in peti length (ielding periods from 16 1935) or chythmiciy pt tn lla tr mRNA and the or ‘esponding proteins are shythmically expressed in ‘Sly fei and by egypt has of enpretion, ‘els and interlocked lope the lod jee proceeds ina robul nner (GENERATION OF THE CECILATON, The proges ofthe Draspielock eyecan be flowed fom the mide ‘ofthe day (Figure723) At thstime most ofthe PER {nd TIM protein nthe el ha been degraded ep {net RNA loves are beginning oie proces tat tl ake about 10 ho peak The transcription factors ELK and C¥C work together ina hetrodimeri or ex the CCC) hind to tes called Esboxes within the Promoters of ter in, and o gen. Tike the WCC in Nesp, the CCCs a rans ‘sonal actator hat dives ienianshythm of rn serpion ls target genes. The areca produce Sad are translated int the commesponcing proteins. VRL foves to the nucleus and reduces CL expression, While PER i in the eytoplsm tis phosphorylated by (Ck2and DBT. This phosphorylation makes PER sus- ‘ope o degradation thereafter unless ts asociated With the TIM roti. Because TIM takes some time © sccumat PERacumplton is delayed an ny ‘transcription of er and in, and PER-TIM omnes (nd kiarekindcarbreseciteratss Sele begins ine SSeicocmchoceeinn Eugen sone ofthe gsi the feedback op ht giver othe Tong (approximately 24h cyl gt “Also he cytoplas, the SCG kinase phosphory- Inte TIM, probably aided by CK2 Phosphorylation of TIM promates TIN's acess othe nucleus, and ths ‘TIM and PER hep each oier nd DBT ito the mace 1 This delay in ule accumulation is another agi the cle Gradually, by early evening, PER and TIM. proteins apper, they heterodimerize and enter the Fuels, Once inthe mucus, TIM gradually dsappens from the comples, and PER Sings abost 9 gran) ‘preston of por an in ranacription. ns ner im ilar to the repression of WCC activity by FRQ In ‘Newrospaa, His key that PER, or possibly she PER-TIM complex, vets tis negative alin the lock cele reducing or preventing the ably ofthe CC tobind tothe yerand in prometes.Inany ease, before ‘mdright, CCC activity has detned fits lowest evel jst a¢ PER level ee o their maxim, 0 together these proces tur down the expresso ofboth Se por dhe genes Ath poat inthe evening PER negatively fet: ing OCC attvity in thence, Without thi erip- tonal activator to deve more mRNAs, er ad ‘in uanscpt evel begin to decline, although ongoing, "Mansation ofthe emaiing mRNAs allows PER and TIM protein levels to contin to i for several ursMolecular Biology of Crcadsn Pacemaker Systems 239 Thus per and tn mNA levels peak inthe early to imidevening about &to 6h Blorethe peak of PER and ‘TiMtinthe mid tolae evening. Also at this ime, another ol of PER in the eye ser: PER (or posibly the PER-TIM compen) in some ‘way cases increased expression of Cito that levels of CLK begin ose even ae phosphoryltion-promoted Inthe cell PER Hocking activation of te per roma. trby the CCC while promoting CLK synthesis to ‘rene the evel ofthe CO. Because CVC aaa n onntatvsly high eves the incesing levels of CLE resulta increasing amount ofthe CLE-CYC cor ples, which sl hel inactive by PER ‘Anaher protein VR alo ats to reinforce his che ‘The CCC binds to E-boxes in thee promote an po motes is synthesis Once made, WRI immediately ‘represses expesion of Cl CLK level fla soon pertim, and ori miRNA levels decline, VR in effect Strengthen the repressvearm ofthe lop beeen ake Alay and minight- Inthe nucleus sta in the cyto plasm ae soon as TIM leaves, DBT phosphorylates ad {hereby destablzes PER, lending to PER's precipitous ‘urmoves As PER degraded, PER-promotd sya OFCLK soon balanced by CLK dpredaton, and CLK levels peak just after dawn near to wen PER levels drop the lowest pine "Te overall eet sa wave of CLK and CCC atv ty created by the juxtaposition of PER-promoted CLK Syhess and te blockage of CC activation. This igh ‘eansripton activating ctv creates sharp tea ‘Son t nit the next eycle a to maintain # robust smpltae nthe fondbsck lop. The Drosoptila clock is reset through lightpro- ‘moted protein degradations" ‘The siadian system n Dos ca be synchronized swith the day-night eye Gough ecanatns hat can ‘etect sal changes in the ambient conditions ether bye pulses of light or small step In lempesatuse Although temperature resetting mechanism a tl tein elucidates, ight is onersoo to eet te cock though a mechanism woling igh promot mover ‘ofthe TIM protein, Drop uses severe thodopein nd favin-based photoneeptrs to det light Laws of ithe opsin basa receptors stl permis entainment, butitates 1 t'days longer to each a sabe paw, “The principal and celhautonomeus photoreceptor forthe clock appeats tobe cyptochrme (CRY) tht, ‘es. ain att chromophore t confer ight ene tyonthe lace sys nthe trl neurone inthe bin ‘01 Drspll, ths CRY hes no role inte cor osclator loop. The brain clok calls are he te of he dominant ‘Pacemaker for behaviocalviyhma, and they can also receive information about ambien ight as tected by ‘hedopin sd phoceporin he compound ye nda epson otc Ge Cp 3) Tken tetera cain enn flag an ‘cite amin to alinperiad ight ark yen Set norton igh tater ne oe Otcrnal ans, Spealation nhs anal sty tescprt cc anton om ght peep a smac West and went elapse a eine ee The mechan tieough which CRY rst the esp clok repens peng synmetywih ‘Somethin sled in ent whch ig indueson ff pay acento Br, PER tnd Tires peck angi and beg aetna {ication of ying afro chartered yew icrcnatbth poi iiss pecan through CRY block the sy of FERCTIN ad ently seater oN. Bacane TM Seren peer 8 om peep sig FER the even th ed tt eget feo TMG Insnansef PR CRY oral pnt bth ie ‘Sonne tnd the mc nui with Salewrdepenent nunner Whe CY bcs ‘Tit Seow modied og tachment oft ‘or pain ben ewig oF “ivi asto sche deena tn pte some Beier sytneins hy carers fo iG ora PER ph meine degen ot theprotisinte eters TM ae PER ‘ho dcloying the phe back tothe previous ny Savery light prooteddoradtion of TM and Pts ight soo rae revel bu advance thedck inthe rete “Th tll a a tes inthe aul nang the eg anne, gas a arty ayer ane daca tht an un geen fe Cpr HA ofthese peripheral cla arate sey Showgh €hemediat TM nove ness sone ne cols CRY ay hve keeping ne ‘yond jut ening ake Altnough the expats Cin mot ise cle den ints Te fous veered CRY nn cock mata on tRdowenacviy enews seacome sre Single-Gene Mutations Have Been Used to Describe a Circadian Oscillator in Mammals One particu informative singl-gene mutant Sas Bentied a hanatersa 8 Intec of lll and molecular complexity, mam ‘allan cain syst ea nae inc {y provdes scarves oon wc to view te gece ‘lacomples phenotype atts fullest The maninalisn ‘readan syst onconpasees mip ayers cou240 Chapter 7 pling, Pacemaers sch ashe SCN, heat ver and kid Fey ae clockshop of coupled osilatrs, ane the SCN is coupled nal these peripheral esilators. Coupling isalo revealed ia these effects trough whi ou pts cach at locomotor sty can ee Back toe fade though the ation of proteins As areal gone {Eimuttions can fet the cian aystem as hole Infact many mation ae known that fst hyo in telatvely minor ways, but these are stil itil Inverpet du tote complex ofthe system. Pechaps Fees son the tations that have had te greatest influence onthe understanding of ceaian blog in ‘mammals are single gene mutations tat have ebust tft on input oro he coe oct as was the case ‘ithsimpler ystems of Drs end Navesoe. "The original mammalian single-gene clock mutant, tay sre 36a spontaneous mutation not inthe mouse brie mama tat favo forceadian physio gia veacarch the golden hamster Maser aur tHe This mutant appeated biden, ina shipment of mst rm supply house, and is character Was ‘evealed during » preliminary sreerig on FUNRiNg ‘shes in preparation fora diferent experiment Figure 728) The mutation was diacovered. because of a8 trsually early onset facto acy under an CD Cele and a shor 22 eee unaing period inthe rg a heerozygoe(au/+) nid Breeding lea Fomor ge a) tat ada very short 20 period length Te lick of cles genetic information about thehamter made the gene very dificult clone in {ly but fev mmecitely provided am exceptional for dissection of physniogial apes of the Ramtec dian system ee Chapter 3) Elects ofthe matation nde runing ontalod pry thecrendon dock oueyare Cok) on entrtnment and activity were noted and ase to ‘develop model fo engalnment and for circadian gat. {igo he roden’setousecle Transplantation of SCN {ee fom ani matant os wipe donor provided inconovertble evidence hat supported he aia the SCN ss the dominant pacemaker a cntol of lcomo foraetivity chythms one Figure 819). The relatively rapid recovery of shyhns in wansplant recipients ‘eves tong role for humeral signals rhythm ‘utp om the SCN, The tn ls demons tht ‘rulpe independent pacerskers could unt same train The mutation wa shoven to ale! ao only pe ff Tength bt ano temperature compensation tn a pecpherl dock thereby connecting tace properties in he mammal a they had been condecied model tem Theta astant hasbeen irvmeneey important in ‘oth physologcl and genetic approaches It demon Staal that mammalian ereaan systems coal be i Sect though the elation and aay of ingle gene ‘mutants eta in model ystems The central clockworks invertebrates is best Understood in house mice" Prof the value ofthis approach came with the iden feaion of Clack, the fst gene associated withthe ‘moleclas mechanism of he clock ms mammal [a3 andar forwatd genic screen (igure 725, the 25 ‘mouse tobe examines displayed 21h lengthening of potion action fo this hk of luck, the ack mt fon was fortuitous for another resaon: Because it showed up ina heterozygote, the mutation was ot recesve a are he cornmonlose-a-uncton mttons that typically ase rom mutgeneses stead the Clock mulation wae gente variant f mich rare sor eh ‘rakes an altered ba stil factional product: Breeding Pised homonygote lncClck progeny tat played Eninital perid length 3 longe han i type, but thot soon graded int setiythmiity in DD contions se Figure 7258.0). The gene wa genetically mapped tochrmosome’ na molecule ture fore that xd any vel get as am he se oe PO ‘id an the gene was cloned Confmaton often tty came in two ways. A very ange agment of DNA ontaiting the id ype gene wat Fest placed nt the fenome ofthe tant and une to str the wild-ype [Phenotype Subsequently, DNA sequencing reveled 3 ingle be pair change that made the CLOCK protein ‘Shore Sequencing showed that CLOCK was 4 DNA birding PAS doraln-conaining poten. The sequence ‘wm silat the PAS proteins sendy associate with ‘lock, Desphila PER protein and WC-1 and C28 Nevo and CLOCK wrasse shown to ave a fa tion asa stato analogous to WC. Conservation of‘Molecule Biology of Circadian Pacemaker Systems structure and function ar important clues fr under- ‘anding the molecule logy ofverebrate clock, ‘Sebsoquen progress in he molecular cssection of ‘the core mammalian clock Ras been api die 9 the sent Ase fmm nan sequences chronabilogista have been able fo find sence homelogs to many cock proteins kw fm model ystems, especially Dspii- Design principles ‘Scena model ystems ate conserved inthe seem of verebrate radian feedback lps. These nlide the trancripson and tarslaton- towed feedback oop with Is resultingshythms in lock component RNAS and proteins and tht use of PAS-PAS heterodimers a pos an egress cp Sun nase snd ernan ese Seeeae aint omy oa yeas SETLNICRS SION ate tse Heme ge inept a os SMTA ia ene eee ewe Tapa poe tagh tiny 0 pine oyna cea sage toca ‘muantcopy of hs eve, the estos atic lang per Vee tahini Fe seers hence tive elements in the lop, Other conserved features se the appearance of interlocked oops which negative ‘lements promote the expression of postive element dn te ipertane a phosphorylation a regs lock Protein tumover and thereby determine prod length Rapid progress has been made in identiying Components of vertebrate circadian feedback oopotrarc The vertebrate circadian feedhack loops ae based on twanscription end warslaton and they employ 3 het. trimer of PAS domain-consining proteins ating as ‘transcriptional activator, wel aa family of PER proteins and CRY proteins as negative elements. In Edition conserved design principles the molecular bbology of verse cieaianorilstors revels pl cation of several locators cifferent roles for sme, Sand possible loss of oles for others. This duplication fn ecupiction of components has added reat the complesity ofthe vertebrate clock, Componeats Jenown tn play important olesin verlbratecracan cullator are the BMALI and CLOCK proteins to ‘orthologs of the Drwophia PER protein (PERI and PER2) and two eeyptochromes, CRY and CRY? Rhyehimety further promoted by csc kinase 1 (CKi) as sean in Dsplead Newraso although i mammals to similar forme ofthis Kise, CK ad perform this unton A transcriptional eprescy REV Etta complete the lit of known components Athi protein with an amino sci sequence ike PER, PERS, {uss but may be involves che in outpt- A protein ‘Sarto TIM is found in the SCN, but no ole can Be {ascribed toi yet. Although the CRY proteins have dine acid Sequences similar to those Found in model systems ther primary functons in the clock app to ‘evomenhat diferent Figure 729), The oes of BML242 Chapter 7 Neg ibe hee (mney FIGURE 725 Molecule components of the mouse cack ‘Rerun uy cn led by taking he ceca ‘Shes och specie moar even he ces Irorkocur (CT 12 fag eves BAT potent Echmion COC uA HC An ann (Croptevetot fr Reverb, and Cry MRNAS me fu the copending pros pet (CT) REV ‘Movoto he teu apres Sl expresion PERand Ri cut tan fom le ace hy ay Dest Kinin ray een, Pr tl ary Bolte PERRY oer ck BCC tip (CT) Are eo SC ei fe Rea Endy INA ees ae Svan a ere ore ‘Soar Rinne poi PERG peed eae ofa iA CD Tomar ihe BER-cty-CR ry compere thee tSrencinteroacpon the Pr Reverie nd ‘Srehiate te oye Aw lsat an era er SS ie een ‘moran, rom tse inthe epee whch happen ihe open paseo the leg. coukcontoled fre ‘Eee Bias Tsar i and epperand and CLOCK, however acting asthe PAS-PAS het ‘rodimers activator of tansripion, seem tobe fully ‘onserved. ln Figure 72 they ave the BMAL-CLOCK fommpln or BC, the standawd PAS-PASheterdime of fukaryticeicadian lek oops. BCC activates expres Son o the Per Cry, and Reverb genes, whieh then encode proteins that elt in fndback to block thee ‘own expression. The REV-ERBat prota binds quickly tothe Ball promote to repress expression PER ard (Ca tgethes a complex wit CK protein, regulate teh other intaellulae location while blocking the city of the BCC. In rents, mutations showing si antve effect on shythmicty are Knoven for Cle, Bal Por er Cry, Ce eer nd Cee, where loss of Berd has only minor elects on ehytonily (igure 727. “The prot of Cr? and Cry2 appear tobe some ‘extent redundant in tat me missing just one of these have only slighly altered clocks, but mize lacking both ave ode (igure 723). a haan, vidas bear ‘ig pount mulation ner? sling inte production fan altered PERZprotcin display ar advanced circa Sn phase of sakering and acivty This characte Is what wold be expected from the work in model s- tems forn entrained lock having a shoe inhrent ps ‘length (oce Chapter 3) and it humans this i mani- feted as advanced sleep phase syndrome, or ASPS see Chapter10, Lib and CKie phosphoryite the PER proteins, leading to their degradation. A spontancoss mutation {hs Kas in he mater nthe a pee) leads shat tening ofthe rhythm's period. Rhythmic expression of Some cockcompones was seen o model ystems and Salo seen in vertebrates mRNA ang fom the Per {erm ial, Rea, nl the Cy genes, ss el eer omesponding pots, arrhythmia expres in ‘Sally fashion ae Figure 728) By appropriate phasing expression, delays, and interlocked ops, the lockMolecular Biology of Circadian Pacemaker Systems 243 Sruuprumansacenor reason mene ema ee edie forthe numb of ays sheen te ‘ner etn mi ail te showed we yt 2D. BAe nba mate shown nee sey ye ‘Speman nf tah fr decent mean bosseysens $n dre (DD) ln ‘ct the con From Bare 201) cycle proceeds in robust mannet, However, there ‘pear to be subtle changes inthe way the loops ae ssserble. Thre are some derences song diferent wertebrate system shaded, but becuse the mouse ppents to be gn model foramen and me eae Fave been described nie meane (nding de arout mutton) the mouse oscllabr willbe described i. (GENERATION OF THE OSCILLATION. The progres ofthe ‘lock eye in el within thesupeachinsmatie nucleus ‘ofa mouse cane felled frm mideveningt seat Fight (ce Figure 725) At this te of day, most of the PER, REV-ERBu. and CRY proteins have been rece) lograe, and oR tad Cry RNA eves ae ow ‘tare begining oie. ly by the way that te sme processes ae happening a these times ithe human brain, The trnscripion factors SMALL (socalled MOPS on humans, ARNT) and CLOCK work together athe heterodimerie BMALI-CLOCK complex (BCC in Fare 725) Teeter ey bi boxes inthe promates of sarous gees, including Pr er, Reverand probaly the Cy genes i active th arcpion {Graualy by early morng the pro teins begin to be traelated. REV-ERBO Sxpression PERI-PER@ can move to and from the nucleus and frm complexes by phystalyasocating with CRY, CRY2, $e the to forms of CK; these proteins ‘move tothe nceus, This portant ifer $esonappesrs toil taba he po teins ima manner remiisent of that served ith PER ane TIM in Ds. ‘The esl a the interior 3 lag prcbably brought about by the degra dation both PER and CRY proteins cc tate in the nucleus in a codependent manner Once Wr, they each pari it ‘ever cdfeet bt by sow pesaps al [Sr avon that onto ous eying ‘One primary action ie, of cous, 10 book the tansrptonslstvationactity ofthe BCC, ab requted ina negative Feedback lop. Ts second action, PER? romots the xprssian o Bil, thereby ountrng the fect of REVERB repre ‘on, and together PER and CRY case ‘eling mal expression, This neloked postv feedback loops functionally bat fot mechanstilly sma fo the promo: ‘on of Cit expression by PER-TIM in Dros and of WC-Aexpression by FRQ in Neos, In the mouse the BCC-DNA complex has been reported to contain PERD and the CRY proteins, 0 in ‘mammals the negative slmente may exe thei fet Inthe clock eyle by reducing the slit ofthe DNA In Droop or Ntrespara i which the negative ele ‘ments oppose DNA binding by the posite ements tn fry css, by midday, BCC acvaton a dalnng fo Toot evel a PER? an CRY level Together these two effec tr down the expression of tnget genes, Including Reverb The PER prosin ae Becoming Phosphor ated bythe foo o cen Kina (CRIS Sand CKIe), an action tht may influence stability oF intracellular location, Eventually 52 result of inhibited expression, Per, Revert, and Cry transcrip levels gin deli,244 Chapter 7 oo A oP Se ER a sew SSS Be - aa ee wo ae ANN > alge ed NH Fothenalaor tcf works ud how sxting workin inal tena parca, trthin 20 cic cele ae Farle wth he msc treating Non Te veal sce oe son ing on he whole vertebrate forges have been noted efor ee Chapter 3) Bea load gts can be rset by 2 shor-durstion light ple {05 minutes or more) but eset tends lobe slow in that the steady-state phase i shits rarely seen on te fist, day after the weatment Ta Seon the amount of res a fing tends to be sal on he ‘er ofa few hours mer - 28a) Strong pe resting is a nw. oy as cused Chapters) never eee Stemi cnimals ater esngle light tetment. Light seen FROUREZ28 Rng so eA nS nly nd ‘ame nue of wipe mice in LD I2 12 al over 20 hin DD. Sonne ain ‘et though he Molec em me aah es noted 1 Expression ‘nvr and Pine SCN of whey snd ny met icc drt ‘Tyee RNA sbundane oie el ana gh nthe SN oh pe est ‘ow)neiyh taco row) na (hind owl ander y nc tour ony mee etn seme gure neti csp ‘ube ae aye anenemert mse urn DD) ws ed et early im the right causes a hase delay no the previous fay, and ight late a sight renin phase advances to thenextay Further te lght ‘esponse is gad such that Hight a night hos effet, whereas ight during the ay ‘Bor dacion me Cty scle Pom Okamura 1983 Towed some hours lerby «dele in PER, REV-ERB, sand CRY levels. As heze negative elements disappear they lave the BCC to vactivate transription toe then jl aco maintains aust ample he feedback oop Although his desertion lacking some ofthe mechanistic deta aesaben model stems Ut Faveboon sted longer the molecule analisof the ‘mammalian oxilatory system is being studied very Inversely, so ove deta wllbeavalabein the nest fate Light rests the vertebrate clock by activating transcription of negative elements nthe Feedback Toopscknaiess “The genecal mechani trough which gh ots in ver: teats to synctverie internal adn cocks wth the feral emvsonnent is understood only in bros ox lie Ar that ve however tcan be ec undertoo, has elaine ite ete in terms of reating theo ’As noted previously i terms fest acdock with components that peak in the daytine, suchas he osc Isto described sn Neuosyon, pic induction ofthe Components serves well a rest the lock, the moe ‘nd very ikly also in people ight i detected by spe ‘alized ganglion cals inthe etn (see Chaps 6 and 11), probably through the se of melanopin a igh sercng protin neti the CRY prot eh are the eset enaors fightin Cros, apparelly hore ths cole in vertbeate light sensing with ‘melanopsin When igh also etna, is detected Uy rode and cones ofthe normal visal system and by specialized cwomophores(pobibly metanopsin and {yplochromes) in ganglion els. An ation potentials triggered snd cared nthe reiniypotnamictrct O the SCN in the ypotalame. There results in release (the neurotansrites haanate and pty odeny- Intecylase seating pepside aso known as PACATE These chemi are deta by several receptors thatMolecular Biology of Circadian Pacemaker Systems 245 ae changes inthe ionic envzonment within the SCN ‘rewron. On ofthese acts oles intact cal ‘Sam from soe inthe endoplen etcum SCN cep font Hses that estat cl oa Stent caletum incene stimuloesthe ati of seve Kinases and tite signaling theighs Kise ose (Inatinase sigalg caseae: phphorlton of hi tal protein kinases eiggered by 3 stimulatory event hee the glutmateinduce reece of nescence tum. Te pro epresnting the al ras the ca Efe then phosphorylate, and thrby activate, these lcd prtcn Ni ee ede a al he {opie ofthe ato Kinase can aon moles of i tinct hed Kinase. Braise singh enzyme city ‘api and catalytic fo phosphonate macy target protein Kinases ech ste, te narizaton of en es Enusubstcesintoaeascdecleaty wil ina pe ampiifcton and transmis ofa sgl othe uta Step) Activtion of Kinase cescades cn hove muliple tects Light at broadly to aetrate an acute espore pray SCN calls thereby premoting te expression numerous ges lading so genes: Uist the ens cascade conceit changesin he stadt of the chromatin, the complex of proteins and DNA that ‘mas upthechromostneswithnthe ius One ofthe ‘Phosphor aga puters = fe CAM? responce de ‘ment binding protein (CREB) a potin ha when sc ‘ae wil bind to specie DNA sxqncen, CRES bind Insite re ound inthe promotes ofthe Perl and Prd sees Induction of Perlis sen inthe SCN of the bypo Uhalamas within 10 minstes of lh fling on the eyes FAGURE729 Light exposure any tine ving the amount Figure 729). This rapid induction of Pert by light appears tobe he primary similis for lght sting oF the dock The Ped gene howe delayed igh duct, nd the level of Ped and Cry expresion are at afc ‘by light The Pet induction respons, a ll os the ‘ruling clckreseting response iscicadanly gated toa modes degre ae Chapter 8 Light sen at ight resus ina large ndotion on gh eve wares pt Sean nthe md to at day yi ite any dation, The resus hat ight seem a ight est the clock thermal ay, ing deta sae ask ae the subjoctive day haste acute effect onthe lock. The way in which ightinduced PERL acs within ‘he feedback loop to rest the lock ot ally under ‘std. The clock within th SCN cll sree aight Slumules within one cle, soy the ext dy the new lock phase i achieved eventhough tex ce rary ‘lays wariets before behavioral hythns recone Pll eet Ths here wot lg within he SCN, aed Blof the deay in phase ting fanaa’ cocks due to events inthe output pthway conning the ‘oc in the SCN tothe yh in the et of he boy Many animals have evolved peripheral clocks that are distinct rom the central-brain cellar tacks ‘One ofthe many dogeas concerning hyn fll by the wayside inthe ast dase was the notion fat m= ‘cellular organisms ad oly ene central lckthat di tated the immediate behavioral shy nic sn the organisms ae ls Chapter I) Isa we now appre Bee sxc rom the top othe btm of he head though Find SCN (ute rows were sted detect he aroun enna tare