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Ukuvela kwama-microbial parasites kuhilela ukulwa phakathi kokukhethwa kwemvelo, okubangela ukuba izimuncagazi zithuthuke, kanye nokukhukhuleka kofuzo, okubangela ukuba izimuncagazi zilahlekelwe izakhi zofuzo futhi ziqongelele ukuguquka kwesimo esiyingozi.Lapha, ukuze siqonde ukuthi lokhu kuphikisana kwenzeka kanjani esikalini se-macromolecule eyodwa, sichaza isakhiwo se-cryo-EM se-ribosome ye-Encephalitozoon cuniculi, i-eukaryotic organism enolunye lwezakhi zofuzo ezincane kunazo zonke emvelweni.Ukwehliswa ngokwedlulele kwe-rRNA ku-E. cuniculi ribosomes kuhambisana nezinguquko zesakhiwo ezingakaze zibonwe, njengokuvela kwezixhumi ezazingaziwa ngaphambilini ezihlanganisiwe ze-rRNA kanye ne-rRNA ngaphandle kwamaqhuqhuva.Ukwengeza, i-E. cuniculi ribosome yasinda ekulahlekeni kwezingcezwana ze-rRNA namaprotheni ngokuthuthukisa ikhono lokusebenzisa ama-molecule amancane njengokulingisa kwesakhiwo sezingcezu namaphrotheni e-rRNA eyonakele.Sekukonke, sibonisa ukuthi izakhiwo zamangqamuzana okucatshangwa ukuthi zincishisiwe, ziwohloka, futhi zingaphansi koshintsho oluwohlozayo zinezindlela eziningi zokunxephezela ezizigcina zisebenza naphezu kokufinyezwa kwamangqamuzana okwedlulele.
Ngenxa yokuthi amaqembu amaningi ama-microbial parasites anamathuluzi emolekyuli ayingqayizivele okuxhaphaza abasingathi bawo, ngokuvamile kufanele sakhe izindlela zokwelapha ezihlukene zamaqembu ahlukene ezinambuzane1,2.Kodwa-ke, ubufakazi obusha bubonisa ukuthi ezinye izici ze-parasite evolution ziyaguquka futhi zibikezeleka kakhulu, okubonisa isisekelo esingaba khona sokungenelela okubanzi kokwelapha kuma-microbial parasites3,4,5,6,7,8,9.
Umsebenzi wangaphambilini uhlonze inkambiso evamile yokuziphendukela kwemvelo kuma-microbial parasites okuthiwa ukuncipha kwe-genome noma i-genome decay10,11,12,13.Ucwaningo lwamanje lubonisa ukuthi lapho ama-microorganisms eyeka indlela yawo yokuphila yamahhala futhi abe ama-parasites angaphakathi kwe-intracellular (noma ama-endosymbionts), ama-genomes awo abhekana nokushintshashintsha okuhamba kancane kodwa okumangalisayo phakathi nezigidi zeminyaka9,11.Enqubweni eyaziwa ngokuthi ukubola kwe-genome, ama-microbial parasites aqongelela izinguquko ezisusayo eziguqula izakhi zofuzo eziningi ezibalulekile ngaphambili zibe ama-pseudogenes, okuholela ekulahlekeni kwezakhi zofuzo kancane kancane kanye nokuwa kokuguquka14,15.Lokhu kugoqa kungabhubhisa kufika ku-95% wezakhi zofuzo ezintweni ezindala kakhulu ezingaphakathi kweseli uma kuqhathaniswa nezinhlobo ezisondelene eziphila mahhala.Ngakho-ke, ukuvela kwama-parasites e-intracellular kuwukudonswa kwempi phakathi kwamandla amabili aphikisanayo: ukukhethwa kwemvelo kwe-Darwin, okuholela ekuthuthukiseni ama-parasites, nokuwa kwe-genome, ukuphonsa ama-parasites ekukhohlweni.Ukuthi i-parasite yakwazi kanjani ukuphuma kulo mdonsiswano futhi igcine ukusebenza kwesakhiwo samangqamuzana akukacaci.
Nakuba indlela yokubola kwe-genome ingaqondwa ngokugcwele, ibonakala yenzeka ikakhulukazi ngenxa yokukhukhuleka okuvamile kofuzo.Ngenxa yokuthi ama-parasite ahlala kubantu abancane, abangahlali ngokobulili, nabanofuzo olulinganiselwe, abakwazi ukuqeda ngempumelelo ukuguqulwa kwezakhi zofuzo okwenzeka ngezinye izikhathi ngesikhathi sokuphindaphinda kwe-DNA.Lokhu kuholela ekuqongeleleni okungenakuhlehliswa kokuguqulwa kwezakhi zofuzo eziyingozi kanye nokunciphisa i-genome ye-parasite.Ngenxa yalokho, i-parasite ayilahlekelwa kuphela izakhi zofuzo ezingasadingeki ukuze ziphile endaweni ye-intracellular.Ukungakwazi kwezibalo ze-parasite ukuqeda ngempumelelo ukuguqulwa kwezakhi zofuzo ngezikhathi ezithile okubangela ukuthi lezi zinguquko zinqwabelane kulo lonke i-genome, okuhlanganisa nezakhi zazo zofuzo ezibaluleke kakhulu.
Iningi lokuqonda kwethu kwamanje ngokuncishiswa kofuzo kusekelwe kuphela ekuqhathanisweni kokulandelana kofuzo, kunokunaka okuncane ezinguqukweni zama-molecule angempela enza imisebenzi yokunakekela indlu futhi asebenza njengezinjongo zezidakamizwa ezingaba khona.Ucwaningo lokuqhathanisa luye lwabonisa ukuthi umthwalo wokuguqulwa kwe-intracellular microbial ocile ubonakala ubeka amaprotheni nama-nucleic acid ukuthi ahluke futhi ahlanganiswe, okuwenza ancike kakhulu ku-chaperone futhi azwele ukushisa19,20,21,22,23.Ukwengeza, ama-parasites ahlukahlukene-ukuziphendukela kwemvelo okuzimele ngezinye izikhathi okuhlukaniswa iminyaka engaba yizigidi eziyizinkulungwane ezingu-2.5-ahlangabezane nokulahlekelwa okufanayo kwezikhungo zokulawula ikhwalithi ku-protein synthesis5,6 kanye nezindlela zokulungisa i-DNA24.Kodwa-ke, kuncane okwaziwayo ngomthelela wendlela yokuphila ye-intracellular kuzo zonke ezinye izakhiwo zama-macromolecules eselula, okuhlanganisa nokujwayela kwamangqamuzana emthwalweni okhulayo wokuguquka okulimazayo.
Kulo msebenzi, ukuze siqonde kangcono ukuvela kwamaprotheni nama-nucleic acid we-intracellular microorganisms, sinqume ukwakheka kwama-ribosomes we-intracellular parasite Encephalitozoon cuniculi.I-E. cuniculi isilwane esifana nesikhunta seqembu le-microsporidia eyisinambuzane enama-eukaryotic genomes amancane ngokungavamile futhi ngenxa yalokho asetshenziswa njengezinto eziphilayo eziyimodeli ukufunda ukubola kwe-genome25,26,27,28,29,30.Muva nje, isakhiwo se-cryo-EM ribosome sinqunyelwe ama-genomes ancishiswe ngokumaphakathi we-Microsporidia, i-Paranosema locustae, ne-Vairimorpha necatrix31,32 (~3.2 Mb genome).Lezi zakhiwo ziphakamisa ukuthi ukulahlekelwa okuthile kokukhulisa i-rRNA kunxeshezelwa ngokwakhiwa kokuxhumana okusha phakathi kwamaprotheni e-ribosomal angomakhelwane noma ukutholwa kwamaprotheni amasha we-msL131,32 ribosomal.Izinhlobo ze-Encephalitozoon (i-genome ~ 2.5 million bp), kanye nesihlobo sazo esiseduze kakhulu i-Ordospora, zibonisa izinga eliphezulu lokuncishiswa kwe-genome kuma-eukaryote - zinezakhi zofuzo ze-protein-coding ezingaphansi kuka-2000, futhi kulindeleke ukuthi ama-ribosomes azo awanazo nje kuphela izingcezu zokunwebeka ze-rRNA (i-rRNA) nama-ribosome ama-ribosome ama-ribosome amane amaprotheni e-ribosomal ngenxa yokuntula kwawo ama-homologue ku-E. cuniculi genome26,27,28.Ngakho-ke, siphethe ngokuthi i-E. cuniculi ribosome ingadalula amasu ayengaziwa ngaphambili okujwayela amangqamuzana ekuboleni kofuzo.
Isakhiwo sethu se-cryo-EM simelela i-eukaryotic cytoplasmic ribosome encane kunazo zonke okufanele ibonakale futhi inikeza ukuqonda kokuthi izinga lokugcina lokunciphisa i-genome lithinta kanjani ukwakheka, ukuhlangana, nokuvela kwemishini yamangqamuzana eyingxenye yeseli.Sithole ukuthi i-E. cuniculi ribosome yephula izimiso eziningi ezilondolozwe kabanzi zokugoqa i-RNA nokuhlanganisa i-ribosome, futhi sathola iphrotheni entsha ye-ribosomal eyayingaziwa ngaphambili.Ngokungalindelekile, sibonisa ukuthi i-microsporidia ribosomes iguqule ikhono lokuhlanganisa ama-molecule amancane, futhi sicabanga ukuthi ukuncishiswa kwe-rRNA namaprotheni kubangela ukuqanjwa kokuziphendukela kwemvelo okungase kunikeze izimfanelo eziwusizo ku-ribosome.
Ukuze sithuthukise ukuqonda kwethu ngokuvela kwamaprotheni nama-nucleic acid ezinto eziphilayo ezingaphakathi kweseli, sinqume ukuhlukanisa izinhlamvu ze-E. cuniculi kumasiko amangqamuzana ezincelisayo ezincelisayo ukuze kuhlanzwe ama-ribosomes awo futhi sinqume ukwakheka kwalawa ma-ribosome.Kunzima ukuthola inani elikhulu le-microsporidia ye-parasitic ngoba i-microsporidia ayikwazi ukukhuliswa endaweni yezakhi.Kunalokho, ziyakhula futhi zizalane kuphela ngaphakathi kwengqamuzana elibambayo.Ngakho-ke, ukuze sithole i-E. cuniculi biomass yokuhlanzwa kwe-ribosome, sathelela umugqa wamaseli ezinso ezincelisayo i-RK13 ngezinhlamvu ze-E. cuniculi futhi sathuthukisa lawa maseli anesifo amasonto ambalwa ukuze sivumele i-E. cuniculi ukuthi ikhule futhi iphindaphindeke.Sisebenzisa i-monolayer yeseli ethelelekile cishe ingxenye yemitha-skwele, sakwazi ukuhlanza cishe u-300 mg wezinhlamvu ze-Microsporidia futhi sizisebenzise ukuze sihlukanise ama-ribosomes.Sabe sesiphazamisa izinhlayiya ezihlanziwe ngobuhlalu bengilazi futhi sahlukanisa ama-ribosomes angcolile sisebenzisa i-stepwise polyethylene glycol fractionation yama-lysates.Lokhu kusivumele ukuthi sithole cishe u-300 µg we-E. cuniculi ribosomes eluhlaza ukuze sihlaziye isakhiwo.
Sibe sesiqoqa izithombe ze-cryo-EM sisebenzisa amasampula e-ribosome abe umphumela futhi sacubungula lezi zithombe sisebenzisa imaski ehambisana ne-ribosomal subunit enkulu, inhloko yeyunithi encane, kanye neyunithi encane.Phakathi nale nqubo, siqoqe izithombe zezinhlayiya ze-ribosomal ezingaba ngu-108,000 kanye nezithombe zekhompyutha ze-cryo-EM ezinesixazululo esingu-2.7 Å (Izibalo Ezingeziwe 1-3).Sibe sesisebenzisa izithombe ze-cryoEM ukuze senze imodeli ye-rRNA, i-ribosomal protein, ne-hibernation factor Mdf1 ehlotshaniswa ne-E. cuniculi ribosomes (Fig. 1a, b).
Isakhiwo se-E. cuniculi ribosome eyinkimbinkimbi ene-hibernation factor Mdf1 (pdb id 7QEP).b Imephu ye-hibernation factor Mdf1 ehlotshaniswa ne-E. cuniculi ribosome.c Imephu yesakhiwo yesibili eqhathanisa i-rRNA etholiwe ezinhlotsheni zeMicrosporidian nezakhiwo ezaziwayo ze-ribosomal.Amaphaneli abonisa indawo yezingcezwana ze-rRNA ezikhulisiwe (ES) nezizinda ezisebenzayo ze-ribosome, okuhlanganisa indawo yokukhipha amakhodi (DC), iluphu ye-sarcinicin (SRL), kanye nesikhungo se-peptidyl transferase (PTC).d Ubuningi be-electron obuhambisana nesikhungo se-peptidyl transferase se-E. cuniculi ribosome siphakamisa ukuthi le sayithi ye-catalytic inesakhiwo esifanayo ku-E. cuniculi parasite kanye nabasingathi bayo, okuhlanganisa i-H. sapiens.e, f Ukuminyana kwama-electron ahambisanayo esikhungo sokukhipha amakhodi (e) kanye nesakhiwo sohlelo sesikhungo sokukhipha amakhodi (f) kubonisa ukuthi u-E. cuniculi unezinsalela U1491 esikhundleni sika-A1491 (E. coli izinombolo) kwamanye ama-eukaryote amaningi.Lolu shintsho luphakamisa ukuthi i-E. cuniculi ingase ibe nozwelo kuma-antibiotics aqondise le sayithi esebenzayo.
Ngokuphambene nezakhiwo ezisungulwe ngaphambilini ze-V. necatrix kanye ne-P. locustae ribosomes (zombili izakhiwo zimelela umndeni we-microsporidia ofanayo Nosematidae futhi zifana kakhulu nezinye), ama-31,32 E. cuniculi ribosomes enza izinqubo eziningi ze-rRNA nokuhlukaniswa kwamaprotheni.Ukushintshaniswa okwengeziwe (Izibalo Ezingeziwe 4-6).Ku-rRNA, izinguquko eziphawuleka kakhulu zihlanganisa ukulahlekelwa okuphelele kwe-25S rRNA fragment ES12L kanye nokuwohloka ngokwengxenye kwama-h39, h41, nama-H18 helices (Fig. 1c, Supplementary Fig. 4).Phakathi kwamaprotheni e-ribosomal, izinguquko eziphawuleka kakhulu zazihlanganisa ukulahlekelwa okuphelele kweprotheyini ye-eS30 kanye nokufinyezwa kwe-eL8, eL13, eL18, eL22, eL29, eL40, uS3, uS9, uS14, uS17, kanye nama-eS7 amaprotheni (Izibalo Ezingeziwe 4, 5).
Ngakho, ukuncishiswa okudlulele kofuzo lwezinhlobo ze-Encephalotozoon/Ordospora kubonakala esakhiweni sazo esiyi-ribosome: I-E. cuniculi ribosomes ibhekana nokulahlekelwa okumangazayo kokuqukethwe kwamaprotheni kuma-eukaryotic cytoplasmic ribosomes angaphansi kokuhlukaniswa kwesakhiwo, futhi awanawo lawo ma-rRNA nezingcezu zamaprotheni ezingaphansi kwesizinda se-eukaryotic esingagcini nje ngokuphila.Isakhiwo se-E. cuniculi ribosome sinikeza imodeli yokuqala ye-molecular yalezi zinguquko futhi yembula izenzakalo zokuziphendukela kwemvelo ezinganakwa yizo zombili i-genomics yokuqhathanisa kanye nezifundo ze-intracellular biomolecular structure (I-Supplementary Fig. 7).Ngezansi, sichaza ngayinye yalezi zenzakalo kanye nomsuka wazo wokuziphendukela kwemvelo okungenzeka kanye nomthelela wazo ongaba khona emsebenzini we-ribosome.
Sibe sesithola ukuthi, ngaphezu kokufinyezwa okukhulu kwe-rRNA, ama-E. cuniculi ribosomes anokwehluka kwe-rRNA kwenye yamasayithi awo asebenzayo.Nakuba isikhungo se-peptidyl transferase se-E. cuniculi ribosome sinesakhiwo esifanayo namanye ama-ribosomes e-eukaryotic (Fig. 1d), isikhungo sokukhipha ikhodi siyahluka ngenxa yokuhlukahluka kokulandelana kwe-nucleotide 1491 (E. coli izinombolo, Fig. 1e, f).Lokhu kuqaphela kubalulekile ngoba indawo yokuqopha ama-ribosome e-eukaryotic ngokuvamile iqukethe izinsalela G1408 ne-A1491 uma kuqhathaniswa nezinsalela zohlobo lwebhaktheriya A1408 kanye ne-G1491.Lokhu kuhlukahluka kusekela ukuzwela okuhlukene kwe-ribosomes yebhaktheriya ne-eukaryotic emndenini we-aminoglycoside wama-antibiotic e-ribosomal namanye ama-molecule amancane aqondise indawo yokukhipha amakhodi.Endaweni yokuqopha i-E. cuniculi ribosome, izinsalela ze-A1491 zathathelwa indawo yi-U1491, okungenzeka kwakha isixhumi esibonakalayo esibophezelayo sama-molecule amancane aqondise le sayithi esebenzayo.Okufanayo kwe-A14901 kukhona nakweminye i-microsporidia efana ne-P. locustae kanye ne-V. necatrix, okuphakamisa ukuthi isakazeke phakathi kwezinhlobo ze-microsporidia (Fig. 1f).
Ngenxa yokuthi amasampula ethu e-E. cuniculi ribosome aye ahlukanisiwe ezinhlamvu ezingasebenzi emzimbeni, sihlole imephu ye-cryo-EM ye-E. cuniculi ukuze kuchazwe ngaphambilini ukubopha i-ribosome ngaphansi kwengcindezi noma izimo zendlala.Izici zokulala 31,32,36,37, 38. Sifanise isakhiwo esisungulwe ngaphambilini se-hibernating ribosome nemephu ye-cryo-EM ye-E. cuniculi ribosome.Ekufakweni kwedokodo, ama-ribosomes e-S. cerevisiae asetshenziswe endaweni eyinkimbinkimbi ene-hibernation factor Stm138, ama-ribosome wesikhonyane ahlanganiswe ne-Lso232 factor, kanye ne-V. necatrix ribosomes eyinkimbinkimbi enezici ze-Mdf1 kanye ne-Mdf231.Ngesikhathi esifanayo, sithole ukuminyana kwe-cryo-EM okuhambisana nesici sokuphumula esingu-Mdf1.Ngokufanayo ne-Mdf1 ebophezela ku-V. necatrix ribosome, i-Mdf1 iphinde ibophezele ku-E. cuniculi ribosome, lapho ivimba khona indawo ka-E ye-ribosome, okungenzeka kusize ukwenza ama-ribosomes atholakale lapho izinhlamvu ze-parasite zingasebenzi emzimbeni lapho ungasebenzi (Umfanekiso 2).).
I-Mdf1 ivimba indawo ka-E ye-ribosome, ebonakala isiza ukwenza i-ribosome ingasebenzi lapho izinhlamvu ze-parasite zingasebenzi.Esakhiweni se-E. cuniculi ribosome, sithole ukuthi i-Mdf1 yakha ukuthintana okungaziwa ngaphambilini ne-L1 ribosome stem, ingxenye ye-ribosome eyenza ukukhululwa kwe-tRNA engasebenzi ku-ribosome ngesikhathi sokuhlanganiswa kwamaprotheni.Laba abathintwayo baphakamisa ukuthi i-Mdf1 ihlukane ne-ribosome isebenzisa indlela efanayo ne-deacetylated tRNA, ihlinzeka ngencazelo engaba khona yokuthi i-ribosome iyisusa kanjani i-Mdf1 ukuze iphinde iqalise ukusebenza kwamaprotheni.
Nokho, isakhiwo sethu siveze ukuthintana okungaziwa phakathi kwe-Mdf1 kanye nomlenze we-ribosome we-L1 (ingxenye ye-ribosome esiza ukukhulula i-tRNA e-deacylated ku-ribosome ngesikhathi sokuhlanganiswa kwamaprotheni).Ikakhulukazi, i-Mdf1 isebenzisa othintana nabo abafanayo njengengxenye yendololwane ye-molecule ye-tRNA deacylated (Fig. 2).Lokhu kumodeliswa kwamangqamuzana okwakungaziwa ngaphambili kwabonisa ukuthi i-Mdf1 iyazihlukanisa ne-ribosome isebenzisa indlela efanayo ne-deacetylated tRNA, echaza ukuthi i-ribosome isusa kanjani lesi sici sokulala ukuze iphinde isebenze kabusha ukuhlanganiswa kwamaprotheni.
Lapho sakha imodeli ye-rRNA, sithole ukuthi i-E. cuniculi ribosome inezingcezu ze-rRNA ezigoqwe ngendlela engavamile, esizibize nge-rRNA ehlanganisiwe (Fig. 3).Kuma-ribosomes ahlanganisa izizinda ezintathu zokuphila, i-rRNA igoqa ibe izakhiwo lapho izisekelo eziningi ze-rRNA zingamabhangqa ayisisekelo futhi zigoqene noma zihlangane namaprotheni e-ribosomal38,39,40.Nokho, kuma-E. cuniculi ribosomes, ama-rRNA abonakala ephula lesi simiso esigoqayo ngokuguqula ezinye zezinhlamvu zazo zibe izifunda ze-rRNA ezingakasombululeki.
Isakhiwo se-H18 25S rRNA helix ku-S. cerevisiae, V. necatrix, ne-E. cuniculi.Ngokuvamile, kuma-ribosomes ahlanganisa izizinda zokuphila ezintathu, lesi sixhumi sihlangana sibe i-RNA helix equkethe izinsalela ezingama-24 kuya kwezingu-34.Ku-Microsporidia, ngokuphambene, lesi sixhumi se-rRNA sincishiswa kancane kancane sibe izixhumanisi ezimbili ezinomucu owodwa ezinothe nge-uridine eziqukethe kuphela izinsalela eziyi-12.Eziningi zalezi zinsalela zichayeka ezincibilikisini.Isibalo sibonisa ukuthi i-microsporidia ye-parasitic ibonakala yephula izimiso ezijwayelekile zokugoqa i-rRNA, lapho izisekelo ze-rRNA zivame ukuhlanganiswa nezinye izisekelo noma ezibandakanyeka ekusebenzelaneni kwe-rRNA-protein.Ku-microsporidia, ezinye izingcezu ze-rRNA zithatha ukugoqa okungekuhle, lapho i-rRNA helix yangaphambili iba ucezu olunomucu owodwa olunwetshwe cishe umugqa oqondile.Ukuba khona kwalezi zifunda ezingavamile kuvumela i-microsporidia rRNA ukuthi ibophe izingcezu ze-rRNA ezikude isebenzisa inani elincane lezisekelo ze-RNA.
Isibonelo esimangalisa kakhulu salolu shintsho lokuziphendukela kwemvelo singabonwa ku-H18 25S rRNA helix (Fig. 3).Ezinhlotsheni ezisuka ku-E. coli kuya kubantu, izisekelo zale rRNA helix ziqukethe ama-nucleotide angu-24-32, akha i-helix engavamile kancane.Ezakhiweni ezihlonzwe ngaphambilini ze-ribosomal ezivela ku-V. necatrix kanye ne-P. locustae,31,32 izisekelo ze-H18 helix azikhohlisiwe ngokwengxenye, kodwa ukubhanqa kwesisekelo se-nucleotide kuyalondolozwa.Nokho, ku-E. cuniculi lesi siqeshana se-rRNA siba izixhumanisi ezimfishane 228UUUGU232 kanye 301UUUUUUUU307.Ngokungafani nezingcezu ezijwayelekile ze-rRNA, lezi zixhumanisi ezinothe nge-uridine azigoqeki noma zithintane kakhulu namaprotheni e-ribosomal.Esikhundleni salokho, bathatha izakhiwo ezivulekile nezivuliwe ngokugcwele lapho imicu ye-rRNA inwetshwa cishe iqonde.Lokhu kuhlanganisa okunwetshiwe kuchaza ukuthi u-E. cuniculi usebenzisa kanjani izisekelo ze-RNA eziyi-12 kuphela ukuze agcwalise igebe elingu-33 Å phakathi kwezinqola ze-H16 ne-H18 rRNA, kuyilapho ezinye izinhlobo zidinga okungenani izisekelo ze-rRNA eziphindwe kabili ukuze zivale igebe.
Ngakho-ke, singabonisa ukuthi, ngokugoqa okungathandeki, i-parasitic microsporidia yenze isu lokuthola inkontileka ngisho nalezo zingxenye ze-rRNA ezihlala zilondolozwe kabanzi kuzo zonke izinhlobo zezilwane ezizindeni ezintathu zokuphila.Ngokusobala, ngokuqongelela uguquko oluguqula amahelisethi e-rRNA abe izixhumanisi ezimfushane ze-poly-U, i-E. cuniculi ingenza izingcezu ze-rRNA ezingajwayelekile eziqukethe ama-nucleotide ambalwa ngangokunokwenzeka ukuze kuhlanganiswe izingcezu ze-distal rRNA.Lokhu kusiza ukuchaza ukuthi i-microsporidia izuze kanjani ukuncishiswa okumangalisayo kwesakhiwo sayo samangqamuzana ngaphandle kokulahlekelwa ubuqotho bayo besakhiwo nokusebenza.
Esinye isici esingavamile se-E. cuniculi rRNA ukubukeka kwe-rRNA ngaphandle kokuqina (Fig. 4).Amaqhuqhuva angama-nucleotide angenawo amapheya ayisisekelo asonteka aphume ku-RNA helix esikhundleni sokucasha kuwo.Ama-protrusions amaningi e-rRNA asebenza njengezinamathiselo zamangqamuzana, esiza ukubopha amaprotheni e-ribosomal aseduze noma ezinye izingcezu ze-rRNA.Amanye amaqhubu asebenza njengamahinge, okuvumela i-rRNA helix ukuthi iguquke futhi igoqe ngokufanele ukuze ikhiqize amaprotheni synthesis 41.
a I-rRNA protrusion (S. cerevisiae numbering) ayikho esakhiweni se-E. cuniculi ribosome, kodwa ikhona kwamanye ama-eukaryote amaningi b E. coli, S. cerevisiae, H. sapiens, kanye ne-E. cuniculi ribosomes yangaphakathi.izimuncagazi azinawo amaqhuqhuva amaningi e-rRNA asendulo, alondolozwe kakhulu.Lokhu kuqina kusimamisa ukwakheka kwe-ribosome;ngakho-ke, ukungabi khona kwabo ku-microsporidia kubonisa ukuzinza okuncishisiwe kokugoqa kwe-rRNA kuma-microsporidia parasites.Ukuqhathanisa neziqu ze-P (iziqu ze-L7/L12 kubhaktheriya) kukhombisa ukuthi ukulahleka kwamaqhubu e-rRNA kwesinye isikhathi kuhambisana nokuvela kwamaqhubu amasha eduze kwamaqhubu alahlekile.I-H42 helix ku-23S/28S rRNA ineqhubu lakudala (U1206 ku-Saccharomyces cerevisiae) elilinganiselwa okungenani libe neminyaka eyizigidi eziyizinkulungwane ezingu-3.5 ngenxa yokuvikelwa kwayo ezizindeni ezintathu zokuphila.Ku-microsporidia, lesi siqhumane siyasuswa.Nokho, iqhubu elisha lavela eduze kweqhubu elilahlekile (A1306 ku-E. cuniculi).
Ngokumangalisayo, sithole ukuthi i-E. cuniculi ribosomes ayinayo iningi lamaqhuqhuva e-rRNA atholakala kwezinye izinhlobo zezilwane, kuhlanganise namaqhubu angaphezu kuka-30 agcinwe kwezinye i-eukaryote (Fig. 4a).Lokhu kulahlekelwa kuqeda ukuxhumana okuningi phakathi kwama-ribosomal subunits kanye nama-rRNA helices aseduze, ngezinye izikhathi kudala ama-void amakhulu angenalutho ngaphakathi kwe-ribosome, okwenza i-E. cuniculi ribosome ibe nezimbotshana uma kuqhathaniswa nama-ribosomes endabuko amaningi (Fig. 4b).Ngokuphawulekayo, sithole ukuthi iningi lalawa maqhuqhuva nalo lalahleka ezakhiweni ezihlonzwe ngaphambili ze-V. necatrix kanye ne-P. locustae ribosome, ezazinganakwa ukuhlaziya kwesakhiwo kwangaphambilini31,32.
Ngezinye izikhathi ukulahlekelwa kwe-rRNA bulges kuhambisana nokuthuthukiswa kwamaqhubu amasha eduze kwe-bulge elahlekile.Isibonelo, i-ribosomal P-stem iqukethe iqhubu le-U1208 (ku-Saccharomyces cerevisiae) elasinda lisuka ku-E. coli liya kubantu ngakho-ke lilinganiselwa eminyakeni eyizigidi eziyizinkulungwane ezingu-3.5 ubudala.Ngesikhathi sokuhlanganiswa kwamaprotheni, lokhu kugoqa kusiza isiqu se-P sinyakaze phakathi kokuhlangana okuvulekile nokuvaliwe ukuze i-ribosome ikwazi ukuthola izici zokuhumusha futhi izilethe kusayithi elisebenzayo.Ku-E. cuniculi ribosomes, lokhu kuqina akukho;kodwa-ke, ukuqina okusha (G883) okutholakala kuphela kumabhangqa esisekelo amathathu kungasiza ekubuyiseleni ukuguquguquka okuhle kwesiqu se-P (Fig. 4c).
Idatha yethu ku-rRNA ngaphandle kwamaqhuqhuva iphakamisa ukuthi ukuncishiswa kwe-rRNA akukhawulelwe ekulahlekeni kwezinto ze-rRNA endaweni ye-ribosome, kodwa kungase futhi kubandakanye i-ribosome nucleus, okudala ukukhubazeka kwe-molecular okukhethekile okungakachazwa kumaseli aphilayo mahhala.izinhlobo eziphilayo ziyabhekwa.
Ngemva kokumodela amaprotheni e-canonical ribosomal kanye ne-rRNA, sithole ukuthi izingxenye ezivamile ze-ribosomal azikwazi ukuchaza izingxenye ezintathu zesithombe se-cryo-EM.Ezimbili zalezi zingcezu zingama-molecule amancane ngosayizi (Fig. 5, Supplementary Fig. 8).Ingxenye yokuqala ifakwe phakathi kwamaprotheni e-ribosomal uL15 kanye ne-eL18 endaweni evame ukuhlala kuyo i-C-terminus ye-eL18, efushaniswa ku-E. cuniculi.Nakuba singenakukwazi ukunquma ukuthi leli ngqamuzana lingubani, ubukhulu nokuma kwalesi siqhingi esiminyana kuchazwa kahle ngokuba khona kwama- molecule e- spermidine.Ukubophezela kwayo ku-ribosome kuzinziswa uguquko oluqondile lwe-microsporidia kumaprotheni e-uL15 (Asp51 ne-Arg56), abonakala enyusa ukuhlobana kwe-ribosome yale molekyuli encane, njengoba evumela i-uL15 ukuthi igoqe i-molecule encane ibe isakhiwo se-ribosomal.Umfanekiso Owengeziwe 2).8, idatha eyengeziwe 1, 2).
I-Cryo-EM imaging ebonisa ukuba khona kwama-nucleotide ngaphandle kwe-ribose eboshwe ku-E. cuniculi ribosome.Ku-E. cuniculi ribosome, le nucleotide ithatha indawo efanayo ne-nucleotide engu-25S rRNA A3186 (izinombolo ze-Saccharomyces cerevisiae) kwamanye ama-ribosome amaningi e-eukaryotic.b Esakhiweni se-ribosomal se-E. cuniculi, le nucleotide itholakala phakathi kwamaprotheni e-ribosomal uL9 kanye ne-eL20, ngaleyo ndlela kusimamise ukuxhumana phakathi kwamaprotheni amabili.cd eL20 ukulandelana kokuhlaziywa kokulondoloza phakathi kwezinhlobo ze-microsporidia.Isihlahla se-phylogenetic sezinhlobo ze-Microsporidia (c) kanye nokulandelana kokulandelana okuningi kweprotheni ye-eL20 (d) kubonisa ukuthi izinsalela ezibopha i-nucleotide F170 kanye ne-K172 zigcinwa ku-Microsporidia evamile, ngaphandle kwe-S. lophii, ngaphandle kwe-branching yokuqala ye-Microsporidia, egcina i-Microsporidia ye-ES39, egcina i-Microsporidia.e Lesi sibalo sibonisa ukuthi izinsalela ezibopha i-nucleotide F170 kanye ne-K172 zikhona kuphela ku-eL20 ye-microsporidia genome encishiswe kakhulu, kodwa hhayi kwamanye ama-eukaryote.Sekukonke, le datha iphakamisa ukuthi ama-ribosomes e-Microsporidian asungule isayithi lokubopha i-nucleotide elibonakala libopha ama-molecule e-AMP futhi liwasebenzisele ukuzinzisa ukusebenzisana kwamaprotheni namaprotheni esakhiweni se-ribosomal.Ukulondolozwa okuphezulu kwalesi siza esibophezelayo eMicrosporidia kanye nokungabikho kwayo kwamanye ama-eukaryote kusikisela ukuthi le sayithi ingase inikeze inzuzo ekhethiwe yokusinda ye-Microsporidia.Ngakho-ke, iphakethe elibopha i-nucleotide ku-microsporidia ribosome alibonakali liyisici esiwohlokayo noma uhlobo lokugcina lokuwohloka kwe-rRNA njengoba kuchazwe ngaphambili, kodwa kunalokho kuwukuqanjwa okusha okuwusizo okuvumela i-microsporidia ribosome ukuthi ibophe ngokuqondile ama-molecule amancane, iwasebenzise njengezingqimba zokwakha zamangqamuzana.amabhlogo wokwakha ama-ribosomes.Lokhu kutholakala kwenza i-microsporidia ribosome kube ukuphela kwe-ribosome eyaziwa ngokusebenzisa i-nucleotide eyodwa njengesakhiwo sayo sokwakha.f Umzila wokuziphendukela kwemvelo okucatshangelwayo osuselwe ekubopheni i-nucleotide.
Isisindo sesibili sesisindo samangqamuzana esiphansi sitholakala kusixhumi esibonakalayo phakathi kwamaprotheni e-ribosomal uL9 kanye ne-eL30 (Fig. 5a).Lesi sixhumanisi sichazwe ngaphambilini esakhiweni se-Saccharomyces cerevisiae ribosome njengendawo ebophayo ye-nucleotide engu-25S ye-rRNA A3186 (ingxenye yesandiso se-ES39L rRNA)38.Kwaboniswa ukuthi kuma-ribosomes e-P. locustae ES39L awohlokayo, lesi sikhombimsebenzisi sibopha i-nucleotide eyodwa engaziwa 31, futhi kucatshangwa ukuthi le nucleotide iwuhlobo lokugcina oluncishisiwe lwe-rRNA, lapho ubude be-rRNA buyizisekelo ezingu-~130-230.I-ES39L yehliselwe ku-nucleotide eyodwa engu-32.43.Izithombe zethu ze-cryo-EM zisekela umbono wokuthi ukuminyana kungachazwa ngama-nucleotide.Kodwa-ke, ukulungiswa okuphezulu kwesakhiwo sethu kubonise ukuthi le nucleotide iyi-molecule ye-extraribosomal, ngokunokwenzeka i-AMP (Fig. 5a, b).
Sibe sesibuza ukuthi ingabe isayithi elibophezelayo le-nucleotide livele ku-E. cuniculi ribosome noma ukuthi lalikhona yini ngaphambilini.Njengoba ukubophezela kwe-nucleotide kuxhunyaniswa kakhulu nezinsalela ze-Phe170 kanye ne-Lys172 ku-eL30 ribosomal protein, sihlole ukulondolozwa kwalezi zinsalela kuma-eukaryote amele ama-4396.Njengasendabeni ye-UL15 ngenhla, sithole ukuthi izinsalela ze-Phe170 ne-Lys172 zigcinwe kakhulu kuphela ku-Microsporidia evamile, kodwa azikho kwamanye ama-eukaryote, kuhlanganise ne-atypical Microsporidia Mitosporidium ne-Amphiamblys, lapho i-ES39L rRNA fragment ingancishisiwe.-e).
Sekuhlangene, le datha isekela umbono wokuthi i-E. cuniculi kanye nenye okungenzeka ukuthi i-canonical microsporidia iguqule ikhono lokuthwebula ngokuyimpumelelo izinombolo ezinkulu zama-metabolite amancane esakhiweni se-ribosome ukuze kunxeshezelwe ukwehla kwamazinga e-rRNA namaprotheni.Ngokwenza kanjalo, baye bahlakulela ikhono eliyingqayizivele lokubopha ama-nucleotide ngaphandle kwe-ribosome, okubonisa ukuthi izakhiwo zamangqamuzana ezinambuzane zinxephezela ngokubamba ama-metabolite amaningi amancane futhi ziwasebenzise njengezilingisi zesakhiwo se-RNA eyonakele nezingcezu zamaprotheni..
Ingxenye yesithathu engalingiswanga yemephu yethu ye-cryo-EM, etholakala kuyunithi enkulu ye-ribosomal.Ukulungiswa okuphakeme kakhulu (2.6 Å) kwemephu yethu kuphakamisa ukuthi lokhu kuminyana kungokwamaprotheni anenhlanganisela eyingqayizivele yezinsalela zeketanga elikhulu, okusivumele ukuthi sihlonze lokhu kuminyana njengeprotheni ye-ribosomal engaziwa ngaphambilini esiyihlonze ngokuthi Yaqanjwa ngokuthi msL2 (Microsporidia- specific protein L2) (izindlela, umfanekiso 6).Ukusesha kwethu kwe-homology kubonise ukuthi i-msL2 ilondolozwe ku-Microsporidia clade yohlobo lwe-Encephaliter ne-Orosporidium, kodwa ayikho kwezinye izinhlobo, kuhlanganise nezinye i-Microsporidia.Esakhiweni se-ribosomal, i-msL2 ithatha igebe elakhiwe ukulahlekelwa kwe-ES31L rRNA eyandisiwe.Kulesi sikhala, i-msL2 isiza ukuzinzisa ukugoqa kwe-rRNA futhi inganxephezela ukulahlekelwa kwe-ES31L (Umfanekiso 6).
Ukuminyana kwe-Electron kanye nemodeli ye-Microsporidia-specific ribosomal protein msL2 etholakala ku-E. cuniculi ribosomes.b Ama-ribosome amaningi e-eukaryotic, okuhlanganisa ne-80S ribosome ye-Saccharomyces cerevisiae, ane-ES19L rRNA amplification elahlekile ezinhlotsheni eziningi ze-Microsporidian.Isakhiwo esasungulwa ngaphambilini se-V. necatrix microsporidia ribosome siphakamisa ukuthi ukulahlekelwa kwe-ES19L kulezi zinambuzane kunxeshezelwa ngokuvela kwephrotheni entsha ye-msL1 ribosomal.Kulolu cwaningo, sithole ukuthi i-E. cuniculi ribosome iphinde yakha iphrotheni yokulingisa i-ribosomal RNA eyengeziwe njengesinxephezelo esibonakalayo sokulahlekelwa kwe-ES19L.Nokho, i-msL2 (okwamanje isichasiselwe njengeprotheyini ecatshangelwayo ECU06_1135) kanye ne-msL1 zinemisuka ehlukile yesakhiwo neyokuziphendukela kwemvelo.c Lokhu kutholakala kokukhiqizwa kwamaphrotheni e-msL1 angahlobene ngokuguqukayo kanye ne-msL2 ribosomal kuphakamisa ukuthi uma ama-ribosome aqongelela ukuguqulwa okulimazayo ku-rRNA yawo, angafinyelela amazinga angakaze abonwe okuhlukahluka kokuqamba ngisho naseqenjini elincane lezinhlobo ezihlobene eduze.Lokhu kutholwa kungasiza ukucacisa umsuka nokuvela kwe-ribosome ye-mitochondrial, eyaziwa nge-rRNA yayo encishiswe kakhulu kanye nokuhlukahluka okungavamile ekubunjweni kwamaprotheni kuzo zonke izinhlobo zezilwane.
Sibe sesiqhathanisa iphrotheni ye-msL2 nephrotheni ye-msL1 echazwe ngaphambilini, okuwukuphela kwephrotheni ye-ribosomal ye-microsporidia-specific etholakala ku-V. necatrix ribosome.Besifuna ukuhlola ukuthi ingabe i-msL1 ne-msL2 zihlobene ngokuziphendukela kwemvelo.Ukuhlaziywa kwethu kubonise ukuthi i-msL1 ne-msL2 zithatha i-cavity efanayo esakhiweni se-ribosomal, kodwa zinezakhiwo ezihlukene eziyinhloko neziphakeme, okubonisa imvelaphi yazo ezimele yokuziphendukela kwemvelo (Fig. 6).Ngakho-ke, ukutholakala kwethu kwe-msL2 kunikeza ubufakazi bokuthi amaqembu ezinhlobo ze-eukaryotic ezihlangene zingashintsha ngokuzimela amaprotheni e-ribosomal ahlukene ukuze kunxeshezelwe ukulahleka kwezingcezwana ze-rRNA.Lokhu kutholakala kuphawuleka ngokuthi iningi lama-ribosomes e-cytoplasmic eukaryotic aqukethe amaprotheni angaguquki, okuhlanganisa nomndeni ofanayo wamaphrotheni e-ribosomal angama-81.Ukubukeka kwe-msL1 kanye ne-msL2 ezigabeni ezahlukahlukene ze-microsporidia ekuphenduleni ekulahlekeni kwezingxenye ze-rRNA ezinwetshiwe kusikisela ukuthi ukucekelwa phansi kwesakhiwo samangqamuzana ezinambuzane kubangela ukuthi izimuncagazi zifune ukuguqulwa kwezakhi zofuzo eziyisinxephezelo, okungagcina kuholele ekutholakaleni kwazo ezinhlobonhlobo zezimuncagazi.izakhiwo.
Ekugcineni, lapho imodeli yethu isiqediwe, saqhathanisa ukwakheka kwe-E. cuniculi ribosome nalokho okwakubikezelwe kusukela ekulandelaneni kofuzo.Amaphrotheni amaningana e-ribosomal, okuhlanganisa i-eL14, eL38, eL41, ne-eS30, ngaphambilini bekucatshangwa ukuthi awatholakali ku-E. cuniculi genome ngenxa yokungabikho okubonakalayo kwama-homologue awo ku-E. cuniculi genome.Ukulahleka kwamaprotheni amaningi e-ribosomal nakho kubikezelwa kwezinye izimuncagazi ezincishisiwe kakhulu ze-intracellular kanye nama-endosymbionts.Isibonelo, nakuba amabhaktheriya amaningi aphilayo aqukethe umndeni ofanayo wamaphrotheni e-ribosomal angu-54, eyi-11 kuphela yale mindeni yamaprotheni enama-homologue atholakalayo ku-genome ngayinye ehlaziywe yebhaktheriya ekhawulelwe ukusingatha.Ukweseka lo mbono, ukulahleka kwamaprotheni e-ribosomal kuye kwabonwa ngokuhlolwa ku-V. necatrix kanye ne-P. locustae microsporidia, engenawo ama-eL38 kanye ne-eL4131,32 amaprotheni.
Nokho, izakhiwo zethu zibonisa ukuthi yi-eL38, eL41, ne-eS30 kuphela ezilahlekile ku-E. cuniculi ribosome.Iphrotheni ye-eL14 igcinwe futhi isakhiwo sethu sabonisa ukuthi kungani le phrotheni ingatholakali ekusesheni kwe-homology (Fig. 7).Ku-E. cuniculi ribosomes, iningi lesizinda sokubopha i-eL14 liyalahleka ngenxa yokucekelwa phansi kwe-rRNA-amplified ES39L.Uma ingekho i-ES39L, i-eL14 yalahlekelwa ingxenye enkulu yesakhiwo sayo sesibili, futhi kuphela u-18% wokulandelana kwe-eL14 owawufana ku-E. cuniculi kanye ne-S. cerevisiae.Lokhu kulondolozwa kokulandelana okungalungile kuyamangaza ngoba ngisho nama-Saccharomyces cerevisiae kanye ne-Homo sapiens—izinto eziphilayo ezavela ngokuhlukana ngeminyaka eyizigidi eziyizinkulungwane ezingu-1.5—zihlanganyela ngaphezu kuka-51% wezinsalela ezifanayo ku-EL14.Lokhu kulahlekelwa okumangalisayo kokongiwa kwemvelo kuchaza ukuthi kungani i-E. cuniculi eL14 njengamanje ichazwa njengeprotheni ebekayo engu-M970_061160 hhayi njengeprotheni ye-eL1427 ribosomal.
kanye ne-Microsporidia ribosome ilahlekelwe isandiso se-ES39L rRNA, esiqede ingxenye ye-eL14 ribosomal protein binding site.Uma ingekho i-ES39L, iphrotheni ye-eL14 microspore ilahlekelwa yisakhiwo sesibili, lapho i-α-helix yangaphambili ebopha i-rRNA idilika ibe yiluphu yobude obuncane.b Ukuhlelwa kokulandelana okuningi kubonisa ukuthi iphrotheni ye-eL14 ilondolozwe kakhulu ezinhlotsheni ze-eukaryotic (ubunikazi bokulandelana obungama-57% phakathi kwemvubelo nama-homologue abantu), kodwa ayilondolozwanga kahle futhi ahlukene ku-microsporidia (lapho okungengaphezu kuka-24% wezinsalela ezifanayo ne-eL14 homologue).kusuka ku-S. cerevisiae noma ku-H. sapiens).Lokhu kulondolozwa kokulandelana okungalungile kanye nokuhluka kwesakhiwo sesibili kuchaza ukuthi kungani i-eL14 homologue ingakaze itholakale ku-E. cuniculi nokuthi kungani le phrotheni kucatshangwa ukuthi ilahleke ku-E. cuniculi.Ngokuphambene, i-E. cuniculi eL14 ngaphambilini yayichasiswe njenge- putative M970_061160 protein.Lokhu kubheka kusikisela ukuthi ukuhlukahluka kwe-microsporidia genome okwamanje kulinganiselwe kakhulu: ezinye izakhi zofuzo okwamanje okucatshangwa ukuthi zilahlekile ku-microsporidia empeleni zigcinwe, nakuba zihlukene kakhulu;esikhundleni salokho, amanye kucatshangwa ukuthi afaka amakhodi kuzakhi zofuzo ze-microsporidia zamaprotheni aqondene nezikelemu (isb, iphrotheni ecatshangelwayo engu-M970_061160) empeleni amakhodi amaprotheni ahluke kakhulu atholakala kwamanye ama-eukaryote.
Lokhu okutholakele kusikisela ukuthi i-rRNA denaturation ingaholela ekulahlekelweni okumangazayo kokulondolozwa kokulandelana kumaprotheni e-ribosomal aseduze, okwenza lawa maprotheni angabonakali ekusesheni kwe-homology.Ngakho, singase silinganisele ngokweqile izinga langempela lokuwohloka kwamangqamuzana ezinto eziphilayo ezincane ze-genome, njengoba amanye amaprotheni okucatshangwa ukuthi alahlekile asekhona, nakuba eshintshashintsha kakhulu.
Ama-parasites angawugcina kanjani umsebenzi wemishini yawo yamangqamuzana ngaphansi kwezimo zokunciphisa ngokweqile kwe-genome?Ucwaningo lwethu luphendula lo mbuzo ngokuchaza ukwakheka kwamangqamuzana ayinkimbinkimbi (i-ribosome) ye-E. cuniculi, into ephilayo enolunye lwezakhi zofuzo ze-eukaryotic ezincane kakhulu.
Sekuyiminyaka engaba ngu-20 kwaziwa ukuthi amaprotheni nama-molecule e-RNA kuma-microbial parasites avame ukuhluka kuma-molecule awo angama-homologous ezinhlotsheni eziphilayo zamahhala ngenxa yokuthi azinazo izikhungo zokulawula ikhwalithi, ancishiswa abe ngu-50% wobukhulu bawo kuma-microbes aphilayo, njll.izinguquko eziningi ezilimazayo eziphazamisa ukugoqa nokusebenza.Isibonelo, ama-ribosome ezinto eziphilayo ezincane ze-genome, okuhlanganisa ama-parasites amaningi e-intracellular kanye nama-endosymbionts, kulindeleke ukuthi angabi namaprotheni amaningana e-ribosomal futhi afinyelele ingxenye eyodwa kwezintathu yama-nucleotide e-rRNA uma kuqhathaniswa nezinhlobo eziphilayo ezikhululekile 27, 29, 30, 49. Nokho, indlela la ma-molecule asebenza ngayo ku-mycosymbies eyinhloko ecwaninga ngokuyinhloko, i-mysternomic genomic isala.
Ucwaningo lwethu lubonisa ukuthi ukwakheka kwama-macromolecules kungaveza izici eziningi zokuziphendukela kwemvelo okunzima ukuzikhipha ezifundweni zendabuko zokuqhathanisa ze-genomic zama-parasites e-intracellular kanye nezinye izinto eziphilayo ezivinjelwe umkhosi (I-Supplementary Fig. 7).Isibonelo, isibonelo sephrotheni ye-eL14 sibonisa ukuthi singalinganisa ngokweqile izinga langempela lokuwohloka kwemishini yamangqamuzana ezinhlotsheni zezinambuzane.Ama-encephalic parasites manje kukholakala ukuthi anezinkulungwane zezakhi zofuzo eziqondene ne-microsporidia.Nokho, imiphumela yethu ibonisa ukuthi ezinye zalezi zakhi zofuzo ezibonakala ziqondile empeleni ziyizinhlobonhlobo ezihluke kakhulu zezakhi zofuzo ezivamile kwamanye ama-eukaryote.Ngaphezu kwalokho, isibonelo sephrotheni ye-msL2 sibonisa ukuthi singakunaki kanjani amaprotheni amasha e-ribosomal futhi sikubuke kancane okuqukethwe kwemishini yamangqamuzana eyisinambuzane.Isibonelo samangqamuzana amancane sibonisa ukuthi singaziba kanjani izinto ezintsha ezihlakaniphe kakhulu ezakhiweni zamangqamuzana ezinambuzane ezingawanikeza umsebenzi omusha wezinto eziphilayo.
Ihlanganiswe ndawonye, ​​le miphumela ithuthukisa ukuqonda kwethu umehluko phakathi kwezakhiwo zamangqamuzana ezinto eziphilayo ezikhawulelwe kanye nozakwabo ezintweni eziphilayo ezikhululekile.Sibonisa ukuthi imishini yamangqamuzana, okucatshangwa ukuthi incishisiwe, iwohloka, futhi engaphansi kwezinguquko ezihlukahlukene eziwohlozayo, esikhundleni salokho inesethi yezici zesakhiwo ezishaywe indiva ngendlela ehlelekile.
Ngakolunye uhlangothi, izingcezu ze-rRNA ezingenayo inqwaba nezingcezu ezihlanganisiwe esizitholile kuma-ribosomes e-E. cuniculi ziphakamisa ukuthi ukuncishiswa kwe-genome kungashintsha ngisho nalezo zingxenye zemishini eyisisekelo yamangqamuzana alondolozwe ezizindeni ezintathu zokuphila - ngemva kweminyaka ecishe ibe yizigidi eziyizinkulungwane ezingu-3.5 .ukuziphendukela kwemvelo okuzimele kwezinhlobo zezilwane.
Izingcezu ze-rRNA ezingenaziqhumane nezihlanganisiwe ku-E. cuniculi ribosomes zithakaselwa ngokukhethekile ekukhanyeni kocwaningo lwangaphambilini lwama-molecule e-RNA kubhaktheriya ye-endosymbiotic.Isibonelo, ku-aphid endosymbiont i-Buchnera aphidicola, i-rRNA kanye nama-molecule e-tRNA aboniswe anezakhiwo ezizwela izinga lokushisa ngenxa yokuchema kokwakheka kwe-A+T kanye nengxenye ephezulu yamapheya ayisisekelo angewona ama-canonical angama-20,50.Lezi zinguquko ku-RNA, kanye nezinguquko kuma-molecule amaprotheni, manje zicatshangwa ukuthi zinesibopho sokuthembela ngokweqile kwama-endosymbionts kubalingani kanye nokungakwazi kwe-endosymbionts ukudlulisa ukushisa kwe-21, i-23.Nakuba i-microsporidia rRNA ye-parasitic inezinguquko ezihlukene ngokwesakhiwo, imvelo yalezi zinguquko iphakamisa ukuthi ukunciphisa ukuzinza okushisayo nokuncika okuphezulu kumaprotheni e-chaperone kungase kube izici ezivamile zama-molecule e-RNA ezintweni eziphilayo ezinama-genomes ancishisiwe.
Ngakolunye uhlangothi, izakhiwo zethu zibonisa ukuthi i-microsporidia ye-parasite iye yashintsha ikhono eliyingqayizivele lokumelana ne-rRNA egcinwe ngokubanzi nezingcezu zamaprotheni, ithuthukisa ikhono lokusebenzisa ama-metabolite amancane atholakala kalula njengamalingi esakhiwo se-rRNA ewohlokayo nezingcezu zamaprotheni.Ukuwohloka kwesakhiwo samangqamuzana..Lo mbono usekelwa iqiniso lokuthi ama-molecule amancane anxephezela ukulahleka kwezingcezu zamaprotheni ku-rRNA kanye nama-ribosomes ka-E. cuniculi abopha izinsalela eziqondene ne-microsporidia ku-uL15 kanye ne-eL30 amaprotheni.Lokhu kuphakamisa ukuthi ukubopha ama-molecule amancane kuma-ribosomes kungase kube umkhiqizo wokukhetha okuhle, lapho ukuguqulwa okuqondile kwe-Microsporidia kumaprotheni e-ribosomal kuye kwakhethwa ngenxa yekhono lawo lokwandisa ukuhlangana kwama-ribosomes kuma-molecule amancane, okungase kuholele ezintweni eziphilayo ezisebenza kahle ze-ribosomal.Ukutholwa kuveza ubuhlakani obusha esakhiweni samangqamuzana ezinambuzane ezincane futhi kusinika ukuqonda kangcono ukuthi izakhiwo zamangqamuzana ezinambuzane ziwugcina kanjani umsebenzi wazo naphezu kokuziphendukela kwemvelo okunciphisayo.
Okwamanje, ukuhlonzwa kwala ma- molecule amancane akukacaci.Akucaci ukuthi kungani ukubukeka kwala ma-molecule amancane esakhiweni se-ribosomal kuhluke phakathi kwezinhlobo ze-microsporidia.Ikakhulukazi, akucaci ukuthi kungani ukubophezela kwe-nucleotide kubonakala ku-ribosomes ye-E. cuniculi ne-P. locustae, hhayi kuma-ribosomes we-V. necatrix, naphezu kokuba khona kwensalela ye-F170 ku-eL20 kanye ne-K172 amaprotheni e-V. necatrix.Lokhu kususwa kungase kubangelwe izinsalela ezingu-43 uL6 (ezitholakala eduze kwephakethe elibopha i-nucleotide), okuyi-tyrosine ku-V. necatrix hhayi i-threonine ku-E. cuniculi ne-P. locustae.Iketango lezinhlangothi ezinephunga elimnandi le-Tyr43 lingaphazamisa ukubophezela kwe-nucleotide ngenxa yokunqwabelana kwesteric.Ngaphandle kwalokho, ukususwa okubonakalayo kwe-nucleotide kungase kube ngenxa yokulungiswa okuphansi kwe-cryo-EM imaging, okuvimbela ukumodela kwe-V. necatrix ribosomal fragments.
Ngakolunye uhlangothi, umsebenzi wethu usikisela ukuthi inqubo yokubola kwe-genome ingase ibe amandla okusungula.Ikakhulukazi, ukwakheka kwe-E. cuniculi ribosome kuphakamisa ukuthi ukulahleka kwe-rRNA nezingcezu zamaprotheni ku-microsporidia ribosome kudala ingcindezi yokuziphendukela kwemvelo ekhuthaza izinguquko esakhiweni se-ribosome.Lezi zinhlobonhlobo zenzeka kude nesayithi elisebenzayo le-ribosome futhi zibonakala zisiza ukugcina (noma ukubuyisela) ukuhlanganiswa kwe-ribosome okungahle kuphazanyiswe i-rRNA encishisiwe.Lokhu kusikisela ukuthi ukuqaliswa okusha kwe-microsporidia ribosome kubonakala sengathi kuguquke kwaba isidingo sokuvimbela ukukhukhuleka kofuzo.
Mhlawumbe lokhu kuboniswa kangcono ngokubopha kwe-nucleotide, okungakaze kubonwe kwezinye izinto eziphilayo kuze kube manje.Iqiniso lokuthi izinsalela ezibopha ama-nucleotide zikhona ku-microsporidia evamile, kodwa hhayi kwamanye ama-eukaryote, liphakamisa ukuthi izindawo ezibopha i-nucleotide azizona nje izinsalela ezilindele ukunyamalala, noma indawo yokugcina ye-rRNA okufanele ibuyiselwe esimweni se-nucleotide ngayinye.Esikhundleni salokho, le sayithi ibonakala iyisici esiwusizo okungenzeka ukuthi sivele emizuliswaneni embalwa yokukhetha okuhle.Iziza ezibophezelayo ze-Nucleotide zingase zibe umkhiqizo wokuzikhethela kwemvelo: uma i-ES39L isicekeleke phansi, i-microsporidia iphoqeleka ukuthi ifune isinxephezelo sokubuyisela i-ribosome biogenesis elungile uma ingekho i-ES39L.Njengoba le nucleotide ikwazi ukulingisa ukuxhumana kwamangqamuzana e-A3186 nucleotide ku-ES39L, i-nucleotide molecule iba ibhilidi le-ribosome, ukubopha kwayo okuthuthukiswa nakakhulu ngokuguquguquka kokulandelana kwe-eL30.
Ngokuphathelene nokuguquguquka kwamangqamuzana ezinambuzane ezingaphakathi kwamangqamuzana, ucwaningo lwethu lubonisa ukuthi amandla okukhethwa kwemvelo kukaDarwin kanye nokukhukhuleka kofuzo kokubola kwe-genome akusebenzi ngokuhambisana, kodwa kuyazungeza.Okokuqala, ukukhukhuleka kwezakhi zofuzo kuqeda izici ezibalulekile zama-biomolecule, okwenza isinxephezelo sidingeke kakhulu.Kuphela uma izimuncagazi zanelisa lesi sidingo ngokukhethwa kwemvelo kukaDarwin lapho ama-macromolecules awo eyoba nethuba lokuthuthukisa izici zawo ezihlaba umxhwele kakhulu nezisungula izinto ezintsha.Okubalulekile, ukuvela kwezingosi ezibophezelayo ze-nucleotide ku-E. cuniculi ribosome kuphakamisa ukuthi le phethini yokulahlekelwa-kuya-kuzuzisa yenguquko yamangqamuzana ayigcini nje ngokudambisa ukuguqulwa kwezakhi zofuzo, kodwa ngezinye izikhathi inikeza imisebenzi emisha ngokuphelele kuma-macromolecules ayizinambuzane.
Lo mbono uhambisana nethiyori kaSewell Wright yokulingana ehambayo, ethi uhlelo oluqinile lokuzikhethela kwemvelo lukhawulela ikhono lezinto eziphilayo lokusungula izinto ezintsha51,52,53.Kodwa-ke, uma ukukhukhuleka kwezakhi zofuzo kuphazamisa ukukhetha kwemvelo, lokhu kukhukhuleka kungaveza izinguquko ngokwazo ezingaguquguquki (noma ngisho eziyingozi) kodwa eziholela kwezinye izinguquko ezihlinzeka ngokufaneleka okuphezulu noma umsebenzi omusha webhayoloji.Uhlaka lwethu lusekela lo mbono ngokubonisa ukuthi uhlobo olufanayo lokuguqula okunciphisa ukugoqa nokusebenza kwe-biomolecule kubonakala kuyimbangela eyinhloko yokuthuthukiswa kwayo.Ngokuhambisana nemodeli yokuwina-win yokuziphendukela kwemvelo, ucwaningo lwethu lubonisa ukuthi ukubola kwe-genome, okuvame ukubhekwa njengenqubo ewohlokayo, kubuye kube umshayeli omkhulu wokuqanjwa kabusha, ngezinye izikhathi futhi mhlawumbe ngisho nokuvame ukuvumela ama-macromolecules ukuthi athole imisebenzi emisha ye-parasitic.angazisebenzisa.