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Ukuvela kwezinambuzane ezincane kuhilela ukulwa phakathi kokukhethwa kwemvelo, okubangela ukuba izinambuzane zithuthuke, kanye nokuguquka kwezakhi zofuzo, okubangela ukuthi izinambuzane zilahlekelwe izakhi zofuzo futhi ziqoqe izinguquko eziyingozi. Lapha, ukuze siqonde ukuthi lokhu kumelana kwenzeka kanjani esikalini se-macromolecule eyodwa, sichaza isakhiwo se-cryo-EM se-ribosome ye-Encephalitozoon cuniculi, into ephilayo ye-eukaryotic ene-genome encane kakhulu emvelweni. Ukuncipha okukhulu kwe-rRNA kuma-ribosome e-E. cuniculi kuhambisana nezinguquko zesakhiwo ezingakaze zibonwe, njengokuvela kwezixhumanisi ze-rRNA ezihlanganisiwe ezazingaziwa ngaphambili kanye ne-rRNA ngaphandle kwamaqhubu. Ngaphezu kwalokho, i-ribosome ye-E. cuniculi yasinda ekulahlekelweni kwezingcezu namaprotheni e-rRNA ngokuthuthukisa ikhono lokusebenzisa ama-molecule amancane njengezinto ezilingiswayo zezakhi ze-rRNA eziwohlokile kanye namaprotheni. Sekukonke, sibonisa ukuthi izakhiwo zama-molecule isikhathi eside okwakucatshangwa ukuthi zincishisiwe, ziyawohloka, futhi zingaphansi kwezinguquko eziwohlokayo zinezindlela eziningi zokubuyisela ezizigcina zisebenza naphezu kokufinyela okukhulu kwama-molecule.
Ngenxa yokuthi amaqembu amaningi ezilokazane ezincane anamathuluzi ahlukile e-molecule okusebenzisa izidalwa zazo, sivame ukuthuthukisa izindlela zokwelapha ezahlukene zamaqembu ahlukene ezilokazane ezincane1,2. Kodwa-ke, ubufakazi obusha busikisela ukuthi ezinye izici zokuvela kwezilokazane ziyahlangana futhi ziyabikezelwa kakhulu, okubonisa isisekelo esingaba khona sokungenelela okubanzi kokwelapha ezilokazane ezincane ezincane3,4,5,6,7,8,9.
Umsebenzi wangaphambilini ukhombe umkhuba ovamile wokuziphendukela kwemvelo kuma-parasite ama-microbial obizwa ngokuthi ukunciphisa i-genome noma ukubola kwe-genome10,11,12,13. Ucwaningo lwamanje lubonisa ukuthi lapho ama-microorganism eyeka indlela yawo yokuphila ekhululekile futhi eba ama-parasite angaphakathi kweseli (noma ama-endosymbionts), ama-genome awo adlula kancane kodwa amangalisayo eminyakeni eyizigidi9,11. Enqubweni eyaziwa ngokuthi ukubola kwe-genome, ama-parasite ama-microbial aqoqa izinguquko ezilimazayo eziguqula ama-gene amaningi abalulekile abe ama-pseudogenes, okuholela ekulahlekelweni kancane kancane kwezakhi zofuzo kanye nokuwa kokuguquka14,15. Lokhu kuwa kungabhubhisa kufike ku-95% wama-gene ezinto eziphilayo ezindala kakhulu zangaphakathi kweseli uma kuqhathaniswa nezinhlobo eziphilayo ezikhululekile ezihlobene eduze. Ngakho-ke, ukuvela kwama-parasite angaphakathi kweseli kuyimpi yokulwa phakathi kwamandla amabili aphikisanayo: Ukukhethwa kwemvelo kukaDarwin, okuholela ekuthuthukisweni kwama-parasite, kanye nokuwa kwe-genome, okwenza ama-parasite alibaleke. Ukuthi i-parasite ikwazile kanjani ukuphuma kulo mqhudelwano futhi igcine umsebenzi wesakhiwo sayo sama-molecule akukacaci.
Nakuba indlela yokubola kwe-genome ingaqondakali ngokugcwele, kubonakala sengathi kwenzeka ikakhulukazi ngenxa yokuguquguquka njalo kwezakhi zofuzo. Ngenxa yokuthi ama-parasite ahlala ezindaweni ezincane, ezingahambisani nobulili, futhi ezilinganiselwe ngokwezakhi zofuzo, awakwazi ukuqeda ngempumelelo izinguquko ezilimazayo ezenzeka ngezinye izikhathi ngesikhathi sokuphindaphinda kwe-DNA. Lokhu kuholela ekuqongeleleni okungenakuguqulwa kwezinguquko eziyingozi kanye nokunciphisa i-genome yama-parasite. Ngenxa yalokho, i-parasite ayilahlekelwa nje kuphela izakhi zofuzo ezingasadingeki ukuze isinde endaweni yangaphakathi kweseli. Ukungakwazi kwezinhlobo zama-parasite ukuqeda ngempumelelo izinguquko ezilimazayo ezingavamile okubangela ukuthi lezi zinguquko ziqongeleleke kulo lonke i-genome, kufaka phakathi izakhi zofuzo zazo ezibaluleke kakhulu.
Ukuqonda kwethu okuningi kwamanje ngokunciphisa i-genome kusekelwe kuphela ekuqhathanisweni kokulandelana kwe-genome, ngaphandle kokunaka izinguquko kuma-molecule angempela enza imisebenzi yokunakekela indlu futhi asebenza njengezinhloso zemithi ezingaba khona. Izifundo zokuqhathanisa zikhombisile ukuthi umthwalo wokuguqulwa kwamagciwane okulimazayo ngaphakathi kweseli kubonakala sengathi ubeka amaprotheni nama-nucleic acids engcupheni yokugoqeka nokuhlangana, okwenza ancike kakhulu ku-chaperone futhi abe nomuzwa omkhulu wokushisa19,20,21,22,23. Ngaphezu kwalokho, ama-parasite ahlukahlukene—ukuziphendukela kwemvelo okuzimele ngezinye izikhathi okuhlukaniswe iminyaka engaba yizigidigidi ezingu-2.5—ahlangabezane nokulahlekelwa okufanayo kwezikhungo zokulawula ikhwalithi ekuhlanganisweni kwawo kwamaprotheni5,6 kanye nezindlela zokulungisa i-DNA24. Kodwa-ke, kuncane okwaziwayo ngomthelela wendlela yokuphila yangaphakathi kweseli kuzo zonke ezinye izakhiwo zama-macromolecule eseli, okuhlanganisa nokuzivumelanisa nama-molecule nomthwalo okhulayo wokuguqulwa okulimazayo.
Kulo msebenzi, ukuze siqonde kangcono ukuvela kwamaprotheni nama-nucleic acid ezinto ezincane ezingaphakathi kweseli, sithole isakhiwo sama-ribosome e-parasite yangaphakathi kweseli i-Encephatozoon cuniculi. I-E. cuniculi iyisidalwa esifana nesikhunta esiyingxenye yeqembu lama-microsporidia angavamile ane-genome encane kakhulu ye-eukaryotic futhi ngenxa yalokho asetshenziswa njengezinto eziphilayo eziyisibonelo ukutadisha ukubola kwe-genome25,26,27,28,29,30. Muva nje, isakhiwo se-ribosome se-cryo-EM sitholakale kuma-genome ancishiswe ngokulinganisela e-Microsporidia, i-Paranosema locustae, kanye ne-Vairimorpha necatrix31,32 (~3.2 Mb genome). Lezi zakhiwo ziphakamisa ukuthi ukulahlekelwa okuthile kokukhulisa i-rRNA kukhokhelwa ukuthuthukiswa kokuxhumana okusha phakathi kwamaprotheni e-ribosomal angomakhelwane noma ukutholwa kwamaprotheni e-ribosomal amasha e-msL131,32. Izinhlobo ze-Encephalitozoon (i-genome ~2.5 million bp), kanye ne-Ordospora ehlobene kakhulu, zibonisa izinga eliphezulu lokuncipha kwe-genome kuma-eukaryotes - zinezakhi zofuzo ezingaphansi kuka-2000 ezibhala amaprotheni, futhi kulindeleke ukuthi ama-ribosome azo awagcini nje ngokungabi nezingcezu zokwandiswa kwe-rRNA (izingcezu ze-rRNA ezihlukanisa ama-ribosome e-eukaryotic kuma-ribosome ebhaktheriya) futhi zinamaprotheni amane e-ribosomal ngenxa yokuntuleka kwawo ama-homologues ku-genome ye-E. cuniculi26,27,28. Ngakho-ke, siphethe ngokuthi i-ribosome ye-E. cuniculi ingaveza amasu angaziwa ngaphambilini okuzivumelanisa nama-molecule ekuboleni kwe-genome.
Isakhiwo sethu se-cryo-EM simelela i-ribosome encane kakhulu ye-eukaryotic cytoplasmic okufanele ichazwe futhi sinikeza ukuqonda ukuthi izinga lokugcina lokunciphisa i-genome lithinta kanjani isakhiwo, ukuhlangana, kanye nokuvela kwemishini yama-molecule eyingxenye yeseli. Sithole ukuthi i-ribosome ye-E. cuniculi yephula izimiso eziningi ezigcinwe kabanzi zokugoqwa kwe-RNA kanye nokuhlanganiswa kwe-ribosome, futhi sathola iphrotheni entsha ye-ribosomal, eyayingaziwa ngaphambili. Ngokungalindelekile, sibonisa ukuthi ama-ribosome e-microsporidia athuthukise ikhono lokubopha ama-molecule amancane, futhi sicabanga ukuthi ukunqunywa ku-rRNA namaprotheni kubangela ukusungula izinto ezintsha okungase kunikeze izimfanelo eziwusizo ku-ribosome.
Ukuze sithuthukise ukuqonda kwethu ngokuvela kwamaprotheni nama-nucleic acid ezintweni eziphilayo ezingaphakathi kweseli, sinqume ukuhlukanisa ama-spores e-E. cuniculi emasikweni amangqamuzana ezilwane ezincelisayo ezinegciwane ukuze sihlanze ama-ribosome awo futhi sinqume isakhiwo sala ma-ribosome. Kunzima ukuthola inani elikhulu lama-microsporidia ayingozi ngoba ama-microsporidia awakwazi ukukhuliswa endaweni yezakhamzimba. Esikhundleni salokho, akhula futhi azale kuphela ngaphakathi kweseli elibambayo. Ngakho-ke, ukuze sithole i-biomass ye-E. cuniculi yokuhlanzwa kwe-ribosome, sithelele umugqa weseli lezinso lezilwane ezincelisayo i-RK13 ngama-spores e-E. cuniculi futhi sakhulisa lawa maseli anesifo amasonto amaningana ukuze sivumele i-E. cuniculi ukuthi ikhule futhi yande. Sisebenzisa ungqimba olulodwa lweseli olunegciwane olungaba yisigamu semitha yesikwele, sikwazile ukuhlanza cishe ama-300 mg ama-spores e-Microsporidia futhi siwasebenzise ukuhlukanisa ama-ribosome. Sabe sesiphazamisa ama-spores ahlanzekile ngama-glass beads futhi sahlukanisa ama-ribosome aluhlaza sisebenzisa i-stepwise polyethylene glycol fractionation yama-lysates. Lokhu kusivumele ukuthi sithole cishe ama-µg angu-300 e-E. cuniculi ribosomes angavuthiwe ukuze ahlaziywe ngesakhiwo.
Sabe sesiqoqa izithombe ze-cryo-EM sisebenzisa amasampula e-ribosome aphumayo futhi sacubungula lezi zithombe sisebenzisa imaski ehambisana ne-subunit enkulu ye-ribosomal, ikhanda le-subunit encane, kanye ne-subunit encane. Phakathi nale nqubo, saqoqa izithombe zezinhlayiya ze-ribosomal ezingaba ngu-108,000 kanye nezithombe ze-cryo-EM ezibaliwe ezinesinqumo esingu-2.7 Å (Izithombe Ezingeziwe 1-3). Sabe sesisebenzisa izithombe ze-cryoEM ukwenza imodeli ye-rRNA, i-ribosomal protein, kanye ne-hibernation factor Mdf1 ehlotshaniswa ne-E. cuniculi ribosomes (Isithombe 1a, b).
a Isakhiwo se-ribosome ye-E. cuniculi esiyinkimbinkimbi ene-hibernation factor Mdf1 (pdb id 7QEP). b Imephu ye-hibernation factor Mdf1 ehlotshaniswa ne-ribosome ye-E. cuniculi. c Imephu yesakhiwo sesibili eqhathanisa i-rRNA etholiwe ezinhlotsheni ze-Microsporidian nezakhiwo ze-ribosomal ezaziwayo. Amaphaneli abonisa indawo yezingcezu ze-rRNA ezikhulisiwe (ES) kanye nezindawo ezisebenzayo ze-ribosome, kufaka phakathi indawo yokubhala ikhodi (DC), i-sarcinicin loop (SRL), kanye nesikhungo se-peptidyl transferase (PTC). d Ubuningi be-electron obuhambisana nesikhungo se-peptidyl transferase se-ribosome ye-E. cuniculi busikisela ukuthi le ndawo ye-catalytic inesakhiwo esifanayo ku-parasite ye-E. cuniculi kanye nabasingathi bayo, kufaka phakathi i-H. sapiens. e, f Ubuningi be-electron obuhambisanayo besikhungo sokubhala ikhodi (e) kanye nesakhiwo se-schematic sesikhungo sokubhala ikhodi (f) kubonisa ukuthi i-E. cuniculi inezinsalela ze-U1491 esikhundleni se-A1491 (inombolo ye-E. coli) kwamanye ama-eukaryotes amaningi. Lolu shintsho lubonisa ukuthi i-E. cuniculi ingase izwele kuma-antibiotic ahlasela le ndawo esebenzayo.
Ngokungafani nezakhiwo ezazisungulwe ngaphambilini ze-ribosomes ze-V. necatrix kanye ne-P. locustae (zombili izakhiwo zimelela umndeni ofanayo we-microsporidia i-Nosematidae futhi zifana kakhulu), ama-ribosomes angu-31,32 E. cuniculi adlula ezinqubweni eziningi ze-rRNA kanye nokuqhekeka kwamaprotheni. Ukuguqulwa okuqhubekayo (Izithombe Ezingeziwe 4-6). Ku-rRNA, izinguquko eziphawulekayo kakhulu zazihlanganisa ukulahlekelwa okuphelele kwengxenye ye-25S rRNA fragment ES12L kanye nokuwohloka okuyingxenye kwe-h39, h41, kanye ne-H18 helices (Isithombe 1c, Umfanekiso Ongeziwe 4). Phakathi kwamaprotheni e-ribosomal, izinguquko eziphawulekayo kakhulu zazihlanganisa ukulahlekelwa okuphelele kwephrotheni ye-eS30 kanye nokufinyezwa kwamaprotheni e-eL8, eL13, eL18, eL22, eL29, eL40, uS3, uS9, uS14, uS17, kanye ne-eS7 (Izithombe Ezingeziwe 4, 5).
Ngakho-ke, ukwehla okukhulu kwama-genome ezinhlobo ze-Encephalotozoon/Ordospora kubonakala esakhiweni sazo se-ribosome: Ama-ribosome e-E. cuniculi abhekana nokulahlekelwa okukhulu kokuqukethwe kwamaprotheni kuma-ribosome e-cytoplasmic eukaryotic ngaphansi kokuhlukaniswa kwesakhiwo, futhi awanazo ngisho nalezo zingcezu ze-rRNA kanye namaprotheni ezilondolozwe kabanzi hhayi kuma-eukaryotes kuphela, kodwa nasezindaweni ezintathu zokuphila. Isakhiwo se-ribosome ye-E. cuniculi sinikeza imodeli yokuqala yama-molecule yalezi zinguquko futhi sembula izehlakalo zokuziphendukela kwemvelo ezinganakwa yi-genomics eqhathaniswayo kanye nezifundo zesakhiwo se-biomolecular sangaphakathi kweseli (Isithombe Esingeziwe 7). Ngezansi, sichaza ngasinye salezi zehlakalo kanye nemvelaphi yazo yokuziphendukela kwemvelo kanye nomthelela wazo ongaba khona emsebenzini we-ribosome.
Sabe sesithola ukuthi, ngaphezu kokunqunywa okukhulu kwe-rRNA, ama-ribosome e-E. cuniculi anokwehluka kwe-rRNA kwenye yezindawo zawo ezisebenzayo. Nakuba isikhungo se-peptidyl transferase se-ribosome ye-E. cuniculi sinesakhiwo esifanayo nezinye i-ribosome ze-eukaryotic (Isithombe 1d), isikhungo sokubhala ikhodi siyahluka ngenxa yokwehluka kokulandelana ku-nucleotide 1491 (inombolo ye-E. coli, Isithombe 1e, f). Lokhu kuqaphela kubalulekile ngoba indawo yokubhala ikhodi yama-ribosome e-eukaryotic ngokuvamile iqukethe izinsalela ze-G1408 ne-A1491 uma kuqhathaniswa nezinsalela zohlobo lwebhaktheriya i-A1408 ne-G1491. Lokhu kwehluka kuyisisekelo sokuzwela okuhlukile kwama-ribosome ebhaktheriya ne-eukaryotic emndenini wama-aminoglycoside wama-antibiotic e-ribosomal namanye ama-molecule amancane ahlose indawo yokubhala ikhodi. Endaweni yokubhala ikhodi ye-E. cuniculi ribosome, i-residue A1491 yathathelwa indawo yi-U1491, okungenzeka ukuthi kwakha isikhombimsebenzisi esiyingqayizivele sokubopha sama-molecule amancane aqondise kule ndawo esebenzayo. Uhlobo olufanayo lwe-A14901 lukhona nakwezinye i-microsporidia ezifana ne-P. locustae kanye ne-V. necatrix, okuphakamisa ukuthi lusakazeke kakhulu phakathi kwezinhlobo ze-microsporidia (Isithombe 1f).
Ngenxa yokuthi amasampula ethu e-ribosome e-E. cuniculi ahlukaniswe kuma-spores angasebenzi kahle emzimbeni, sihlole imephu ye-cryo-EM ye-E. cuniculi ukuthola ukubopha kwe-ribosome okuchazwe ngaphambilini ngaphansi kwezimo zokucindezeleka noma zokulamba. Izici ze-hibernation 31,32,36,37, 38. Sifanise isakhiwo esisungulwe ngaphambilini se-ribosome e-hibernating nemephu ye-cryo-EM ye-ribosome ye-E. cuniculi. Ukuze kubekwe idokodo, ama-ribosome e-S. cerevisiae asetshenziswe enkingeni ene-hibernation factor Stm138, ama-ribosome e-loucher enkingeni ene-Lso232 factor, kanye nama-ribosome e-V. necatrix enkingeni ene-Mdf1 kanye ne-Mdf231 factor. Ngesikhathi esifanayo, sithole ubuningi be-cryo-EM obuhambisana ne-rest factor Mdf1. Ngokufanayo nokubopha kwe-Mdf1 ne-ribosome ye-V. necatrix, i-Mdf1 ibopha ne-ribosome ye-E. cuniculi, lapho ivimba khona indawo ye-E ye-ribosome, mhlawumbe isiza ekwenzeni ama-ribosome atholakale lapho ama-spores e-parasite engasebenzi kahle emzimbeni lapho umzimba ungasebenzi (Isithombe 2).
I-Mdf1 ivimba indawo ye-E ye-ribosome, okubonakala sengathi kusiza ekuvimbeleni i-ribosome lapho ama-spores ezinambuzane engasebenzi kahle emzimbeni. Esakhiweni se-ribosome ye-E. cuniculi, sithole ukuthi i-Mdf1 yakha ukuxhumana okungaziwa ngaphambili nesiqu se-ribosome se-L1, ingxenye ye-ribosome esiza ekukhishweni kwe-tRNA e-deacylated kusuka ku-ribosome ngesikhathi sokwenziwa kwamaprotheni. Lokhu kuxhumana kusikisela ukuthi i-Mdf1 iyahlukana ne-ribosome isebenzisa indlela efanayo ne-tRNA e-deacetylated, okunikeza incazelo engaba khona yokuthi i-ribosome isusa kanjani i-Mdf1 ukuze ivuselele ukwenziwa kwamaprotheni.
Kodwa-ke, isakhiwo sethu sembule ukuthintana okungaziwa phakathi kwe-Mdf1 kanye nomlenze we-ribosome we-L1 (ingxenye ye-ribosome esiza ekukhipheni i-tRNA e-deacylated kusuka ku-ribosome ngesikhathi sokwenziwa kwamaprotheni). Ikakhulukazi, i-Mdf1 isebenzisa ukuthintana okufanayo nengxenye yendololwane ye-molecule ye-tRNA e-deacylated (Isithombe 2). Lokhu kumodela kwama-molecule okungaziwa ngaphambilini kubonise ukuthi i-Mdf1 iyahlukana ne-ribosome isebenzisa indlela efanayo ne-tRNA e-deacetylated, okuchaza ukuthi i-ribosome isusa kanjani lesi sici sokulala ukuze ivuselele ukwenziwa kwamaprotheni.
Lapho sakha imodeli ye-rRNA, sithole ukuthi i-ribosome ye-E. cuniculi inezingcezu ze-rRNA ezigoqwe ngendlela engavamile, esizibize ngokuthi i-rRNA ehlanganisiwe (Umfanekiso 3). Kuma-ribosome ahlanganisa izizinda ezintathu zokuphila, i-rRNA igoqeka ibe yizakhiwo lapho iningi le-rRNA lisekela khona noma ligoqeka noma lisebenzisana namaprotheni e-ribosomal38,39,40. Kodwa-ke, kuma-ribosome e-E. cuniculi, ama-rRNA abonakala ephula lesi simiso sokugoqeka ngokuguqula amanye ama-helice awo abe yizifunda ze-rRNA ezingavuleki.
Isakhiwo se-H18 25S rRNA helix ku-S. cerevisiae, V. necatrix, kanye ne-E. cuniculi. Ngokuvamile, kuma-ribosome ahlanganisa izizinda ezintathu zokuphila, le linker ihlangana ibe yi-RNA helix equkethe izinsalela ezingu-24 kuya ku-34. Ku-Microsporidia, ngokuphambene nalokho, le linker ye-rRNA incishiswa kancane kancane ibe yizihlanganisi ezimbili ezine-single-stranded uridine-rich linksers eziqukethe izinsalela ezingu-12 kuphela. Iningi lalezi zinsalela livezwa ezinyibilikisi. Lesi sibalo sibonisa ukuthi i-microsporidia ye-parasitic ibonakala iphula izimiso ezijwayelekile zokugoqwa kwe-rRNA, lapho izisekelo ze-rRNA zivame ukuhlanganiswa nezinye izisekelo noma zihileleke ekusebenzisaneni kwe-rRNA-protein. Ku-microsporidia, ezinye izingcezu ze-rRNA ziba yi-fold engathandeki, lapho i-helix yangaphambili ye-rRNA iba yingcezu ene-stranded eyodwa ende cishe emgqeni oqondile. Ukuba khona kwalezi zindawo ezingavamile kuvumela i-microsporidia rRNA ukuthi ibophe izingcezu ze-rRNA ezikude isebenzisa inani elincane lezisekelo ze-RNA.
Isibonelo esiphawuleka kakhulu salolu shintsho lokuziphendukela kwemvelo singabonakala ku-H18 25S rRNA helix (Isithombe 3). Ezinhlotsheni kusukela ku-E. coli kuya kubantu, izisekelo zale helix ye-rRNA ziqukethe ama-nucleotide angu-24-32, akha i-helix engajwayelekile kancane. Ezinhlakeni ze-ribosomal ezitholakale ngaphambilini ezivela ku-V. necatrix kanye ne-P. locustae,31,32 izisekelo ze-helix ye-H18 azigoqeki ngokwengxenye, kodwa ukuhlangana kwesisekelo se-nucleotide kuyagcinwa. Kodwa-ke, ku-E. cuniculi lesi siqeshana se-rRNA siba yizixhumanisi ezimfushane kakhulu ezingu-228UUUGU232 kanye no-301UUUUUUUUU307. Ngokungafani nezingcezu ezivamile ze-rRNA, lezi zixhumanisi ezicebile nge-uridine azigoqeki noma zenze ukuxhumana okukhulu namaprotheni e-ribosomal. Esikhundleni salokho, zamukela izakhiwo ezivulekile futhi ezivuleke ngokuphelele lapho imicu ye-rRNA inwetshwa khona cishe iqonde. Lokhu kwakheka okunwetshiwe kuchaza indlela i-E. cuniculi esebenzisa ngayo izisekelo ze-RNA ezingu-12 kuphela ukugcwalisa igebe elingu-33 Å phakathi kwama-helice e-H16 ne-H18 rRNA, kuyilapho ezinye izinhlobo zidinga okungenani izisekelo ze-rRNA eziphindwe kabili ukugcwalisa igebe.
Ngakho-ke, singabonisa ukuthi, ngokugoqwa okungathandeki ngamandla, ama-microsporidia aphilayo athuthukise isu lokuncishisa ngisho nalezo zingxenye ze-rRNA ezihlala zilondolozwe kabanzi kuzo zonke izinhlobo ezizindeni ezintathu zokuphila. Ngokusobala, ngokuqongelela izinguquko eziguqula ama-helices e-rRNA abe ama-poly-U linker amafushane, i-E. cuniculi ingakha izingcezu ze-rRNA ezingavamile eziqukethe ama-nucleotide ambalwa ngangokunokwenzeka ukuze kuhlanganiswe izingcezu ze-rRNA ezikude. Lokhu kusiza ukuchaza ukuthi i-microsporidia ifinyelele kanjani ekunciphiseni okukhulu kwesakhiwo sayo esiyisisekelo sama-molecule ngaphandle kokulahlekelwa ubuqotho bayo besakhiwo nokusebenza.
Esinye isici esingavamile se-E. cuniculi rRNA ukubonakala kwe-rRNA ngaphandle kokuqina (Isithombe 4). Ama-bulges angama-nucleotide angenazo izisekelo ezimbili ezisontayo ziphuma ku-RNA helix esikhundleni sokucasha kuyo. Ama-protrusion amaningi e-rRNA asebenza njengezinamathiselo zama-molecule, esiza ukubopha amaprotheni e-ribosomal aseduze noma ezinye izingcezu ze-rRNA. Amanye ama-bulges asebenza njengama-hinge, okuvumela i-rRNA helix ukuthi igobe futhi igoqeke kahle ukuze kukhiqizwe amaprotheni akhiqizayo 41.
a Ukuvela kwe-rRNA (inombolo ye-S. cerevisiae) akukho esakhiweni se-ribosome se-E. cuniculi, kodwa kukhona kwamanye ama-eukaryotes amaningi e-b E. coli, S. cerevisiae, H. sapiens, kanye ne-E. cuniculi internal ribosomes. Ama-parasite awanayo inqwaba yama-rRNA asendulo, agcinwe kahle. Lokhu kuqina kuzinzisa isakhiwo se-ribosome; ngakho-ke, ukungabikho kwawo ku-microsporidia kubonisa ukuqina okunciphile kokugoqeka kwe-rRNA kuma-parasite e-microsporidia. Ukuqhathaniswa neziqu ze-P (iziqu ze-L7/L12 kumabhaktheriya) kukhombisa ukuthi ukulahleka kwama-rRNA bumps ngezinye izikhathi kuhambisana nokuvela kwama-bumps amasha eduze kwama-bumps alahlekile. I-H42 helix ku-23S/28S rRNA ine-bump yasendulo (U1206 ku-Saccharomyces cerevisiae) okulinganiselwa ukuthi ineminyaka okungenani eyizigidigidi ezingu-3.5 ubudala ngenxa yokuvikelwa kwayo ezizindeni ezintathu zokuphila. Ku-microsporidia, leli bump liyasuswa. Kodwa-ke, kwavela iqhubu elisha eduze kweqhubu elilahlekile (i-A1306 ku-E. cuniculi).
Okumangalisayo ukuthi sithole ukuthi ama-ribosome e-E. cuniculi awanawo ama-bulge amaningi e-rRNA atholakala kwezinye izinhlobo, kufaka phakathi ama-bulge angaphezu kuka-30 agcinwe kwezinye i-eukaryotes (Isithombe 4a). Lokhu kulahlekelwa kuqeda ukuxhumana okuningi phakathi kwama-subunit e-ribosomal kanye nama-helices e-rRNA aseduze, ngezinye izikhathi kudala izikhala ezinkulu ezingenalutho ngaphakathi kwe-ribosome, okwenza i-ribosome ye-E. cuniculi ibe nezikhala eziningi uma kuqhathaniswa nama-ribosome endabuko (Isithombe 4b). Okuphawulekayo ukuthi sithole ukuthi iningi lala ma-bulge lalahleka nasezinhlakeni ze-ribosome ze-V. necatrix kanye ne-P. locustae ezazikhonjwe ngaphambilini, ezazinganakwa ukuhlaziywa kwesakhiwo kwangaphambilini31,32.
Ngezinye izikhathi ukulahleka kwamaqhubu e-rRNA kuhambisana nokukhula kwamaqhubu amasha eduze kweqhubu elilahlekile. Isibonelo, isiqu se-ribosomal P siqukethe iqhubu le-U1208 (ku-Saccharomyces cerevisiae) elasinda kusukela ku-E. coli laya kubantu ngakho-ke kulinganiselwa ukuthi lineminyaka eyizigidigidi ezingu-3.5 ubudala. Ngesikhathi sokwenziwa kwamaprotheni, leli qhubu lisiza isiqu se-P ukuthi sihambe phakathi kokwakheka okuvulekile nokuvaliwe ukuze i-ribosome ikwazi ukuthola izinto zokuhumusha futhi izilethe endaweni esebenzayo. Ku-ribosomes ze-E. cuniculi, lokhu kuqina akukho; noma kunjalo, ukuqina okusha (G883) okutholakala kuphela ngamabhangqa amathathu ayisisekelo kungasiza ekubuyiselweni kokuguquguquka okuhle kwesiqu se-P (Isithombe 4c).
Idatha yethu ku-rRNA engenamaqhubu iphakamisa ukuthi ukunciphisa i-rRNA akugcini ekulahlekelweni kwezinto ze-rRNA ebusweni be-ribosome, kodwa kungase futhi kuhilele i-nucleus ye-ribosome, okudala isici samangqamuzana esikhethekile se-parasite esingazange sichazwe kumaseli aphilayo akhululekile. Izinhlobo eziphilayo ziyabonwa.
Ngemva kokwenza amamodeli amaprotheni e-ribosomal kanye ne-rRNA, sithole ukuthi izingxenye ze-ribosomal ezivamile azikwazi ukuchaza izingxenye ezintathu zesithombe se-cryo-EM. Ezimbili zalezi zingcezu zingama-molecule amancane ngobukhulu (Isithombe 5, Umfanekiso Ongeziwe 8). Ingxenye yokuqala ihlukaniswe phakathi kwamaprotheni e-ribosomal uL15 kanye ne-eL18 endaweni evame ukuhlala yi-C-terminus ye-eL18, efinyeziwe ku-E. cuniculi. Nakuba singenakukwazi ukunquma ukuthi iyini le molekyuli, ubukhulu nesimo salesi siqhingi sobuningi sichazwa kahle ngokuba khona kwama-molecule e-spermidine. Ukubopha kwayo ku-ribosome kuzinziswa izinguquko ezithile ze-microsporidia kumaprotheni e-uL15 (Asp51 kanye ne-Arg56), okubonakala kukhulisa ukusondelana kwe-ribosome yale molekyuli encane, njengoba ivumela i-uL15 ukuthi igoqe i-molecule encane ibe yisakhiwo se-ribosomal. Umfanekiso Ongeziwe 2). 8, idatha eyengeziwe 1, 2).
Isithombe se-Cryo-EM esibonisa ukuba khona kwama-nucleotide ngaphandle kwe-ribose eboshelwe kwi-ribosome ye-E. cuniculi. Ku-ribosome ye-E. cuniculi, le nucleotide ithatha indawo efanayo ne-nucleotide ye-25S rRNA A3186 (i-Saccharomyces cerevisiae numbering) kwamanye ama-ribosome amaningi eukaryotic. b Esakhiweni se-ribosomal se-E. cuniculi, le nucleotide itholakala phakathi kwamaprotheni e-ribosomal uL9 kanye ne-eL20, ngaleyo ndlela kuzinzisa ukuxhumana phakathi kwamaprotheni amabili. cd eL20 ukuhlaziywa kokulondolozwa kochungechunge phakathi kwezinhlobo ze-microsporidia. Umuthi we-phylogenetic wezinhlobo ze-Microsporidia (c) kanye nokuhlelwa kwe-multiple sequence kweprotheyini ye-eL20 (d) kubonisa ukuthi izinsalela zokubopha i-nucleotide F170 kanye ne-K172 zilondolozwe ku-Microsporidia ejwayelekile kakhulu, ngaphandle kwe-S. lophii, ngaphandle kwe-Microsporidia yokuqala yamagatsha, egcine isandiso se-ES39L rRNA. Lesi sibalo sibonisa ukuthi izinsalela zokubopha i-nucleotide F170 kanye ne-K172 zikhona kuphela ku-eL20 ye-genome ye-microsporidia encishisiwe kakhulu, kodwa hhayi kwamanye ama-eukaryotes. Sekukonke, le datha iphakamisa ukuthi ama-ribosome e-Microsporidian athuthukise indawo yokubopha i-nucleotide ebonakala ibopha ama-molecule e-AMP futhi iwasebenzisele ukuzinzisa ukusebenzisana kwamaprotheni neprotheyini esakhiweni se-ribosomal. Ukulondolozwa okuphezulu kwale ndawo yokubopha ku-Microsporidia kanye nokungabikho kwayo kwamanye ama-eukaryotes kusikisela ukuthi le ndawo inganikeza inzuzo yokusinda ekhethiwe ye-Microsporidia. Ngakho-ke, isikhwama esibopha i-nucleotide ku-microsporidia ribosome asibonakali siyisici esiwohlokayo noma uhlobo lokugcina lokuwohloka kwe-rRNA njengoba kuchaziwe ngaphambili, kodwa kunalokho kuyindlela entsha ewusizo yokuziphendukela kwemvelo evumela i-microsporidia ribosome ukuthi ibophe ngqo ama-molecule amancane, iwasebenzise njengamabhulokhi okwakha ama-molecule. Lokhu kutholakala kwenza i-microsporidia ribosome ibe yi-ribosome kuphela eyaziwa ngokusebenzisa i-nucleotide eyodwa njengebhulokhi layo lokwakha. f Indlela yokuziphendukela kwemvelo ecatshangelwayo etholakala ekubopheni kwe-nucleotide.
Ubuningi besibili obuphansi besisindo sama-molecule butholakala endaweni yokuxhumana phakathi kwamaprotheni e-ribosomal uL9 kanye ne-eL30 (Isithombe 5a). Lesi sixhumi sachazwa ngaphambilini esakhiweni se-Saccharomyces cerevisiae ribosome njengendawo yokubopha ye-nucleotide engu-25S ye-rRNA A3186 (ingxenye yesandiso se-ES39L rRNA)38. Kwaboniswa ukuthi kuma-ribosome e-P. locustae ES39L agugile, lesi sixhumi sibopha i-nucleotide eyodwa engaziwa engu-31, futhi kucatshangwa ukuthi le nucleotide iwuhlobo lokugcina oluncishisiwe lwe-rRNA, lapho ubude be-rRNA buyizisekelo ezingu-~130-230. I-ES39L yehliswa ibe yi-nucleotide eyodwa engu-32.43. Izithombe zethu ze-cryo-EM zisekela umqondo wokuthi ubuningi bungachazwa yi-nucleotide. Kodwa-ke, ukulungiswa okuphezulu kwesakhiwo sethu kubonise ukuthi le nucleotide iyi-molecule engaphandle kwe-ribosomal, mhlawumbe i-AMP (Isithombe 5a, b).
Sabe sesibuza ukuthi indawo yokubopha i-nucleotide ivele yini ku-ribosome ye-E. cuniculi noma ngabe yayikhona ngaphambilini. Njengoba ukubopha i-nucleotide kuqondiswa kakhulu yizinsalela ze-Phe170 ne-Lys172 kuphrotheni ye-eL30 ribosomal, sihlole ukulondolozwa kwalezi zinsalela kuma-eukaryotes angu-4396 amele. Njengasendabeni ye-uL15 ngenhla, sithole ukuthi izinsalela ze-Phe170 ne-Lys172 zilondolozwe kakhulu kuphela ku-Microsporidia ejwayelekile, kodwa azikho kwamanye ama-eukaryotes, kufaka phakathi i-Microsporidia Mitosporidium ne-Amphiamblys engajwayelekile, lapho ingxenye ye-ES39L rRNA ingancishiswa khona 44, 45, 46 (Isithombe 5c). -e).
Uma zihlanganiswa, le mininingwane isekela umqondo wokuthi i-E. cuniculi kanye nezinye i-canonical microsporidia zithuthukise ikhono lokubamba ngempumelelo inani elikhulu lama-metabolite amancane esakhiweni se-ribosome ukuze kulungiswe ukwehla kwamazinga e-rRNA kanye namaprotheni. Ngokwenza kanjalo, bathuthukise ikhono eliyingqayizivele lokubopha ama-nucleotide ngaphandle kwe-ribosome, okubonisa ukuthi izakhiwo zama-molecule ezidla amagciwane zikhokhela ngokubamba ama-metabolite amancane amaningi futhi ziwasebenzise njengezinto ezilingiswayo zesakhiwo se-RNA ezonakele kanye nezingcezu zamaprotheni.
Ingxenye yesithathu engalingiswanga yemephu yethu ye-cryo-EM, etholakala ku-subunit enkulu ye-ribosomal. Isixazululo esiphezulu (2.6 Å) semephu yethu sibonisa ukuthi lobu buningi bungobamaprotheni anezinhlanganisela ezihlukile zezinsalela ezinkulu ze-side chain, okwasivumela ukuthi sibone lobu buningi njengeprotheni ye-ribosomal engaziwa ngaphambili esiyithole njenge-Yaqanjwa ngokuthi i-msL2 (iprotheni ethile ye-Microsporidia L2) (izindlela, isithombe 6). Ukusesha kwethu kwe-homology kubonise ukuthi i-msL2 igcinwe ku-Microsporidia clade yohlobo lwe-Encephaliter ne-Orosporidium, kodwa ayikho kwezinye izinhlobo, kufaka phakathi ezinye i-Microsporidia. Esakhiweni se-ribosomal, i-msL2 ithatha igebe elakhiwe ukulahleka kwe-ES31L rRNA enwetshiwe. Kulesi sivalo, i-msL2 isiza ukuzinzisa ukugoqeka kwe-rRNA futhi ingakhokhela ukulahleka kwe-ES31L (Isithombe 6).
Ubuningi be-electron kanye nemodeli yeprotheyini ye-ribosomal ethize ye-Microsporidia msL2 etholakala kuma-ribosome e-E. cuniculi. b Ama-ribosome amaningi e-eukaryotic, okuhlanganisa ne-ribosome engu-80S ye-Saccharomyces cerevisiae, amplification ye-ES19L rRNA elahlekile ezinhlotsheni eziningi ze-Microsporidian. Isakhiwo esisungulwe ngaphambilini se-V. necatrix microsporidia ribosome sibonisa ukuthi ukulahlekelwa kwe-ES19L kula ma-parasite kukhokhelwa ukuvela kweprotheyini entsha ye-msL1 ribosomal. Kulolu cwaningo, sithole ukuthi i-E. cuniculi ribosome iphinde yakha iphrotheyini eyengeziwe ye-ribosomal RNA njengesinxephezelo esibonakalayo sokulahlekelwa yi-ES19L. Kodwa-ke, i-msL2 (okwamanje echazwa njengeprotheyini ye-ECU06_1135 ecatshangelwayo) kanye ne-msL1 zinemvelaphi ehlukile yesakhiwo kanye nokuziphendukela kwemvelo. Lokhu kutholakala kokukhiqizwa kwamaprotheni e-ribosomal e-msL1 kanye ne-msL2 angahlobene ngokuziphendukela kwemvelo kusikisela ukuthi uma ama-ribosome eqoqa izinguquko ezilimazayo ku-rRNA yawo, angafinyelela amazinga angakaze abonwe okuhlukahluka kokwakheka ngisho naseqenjini elincane lezinhlobo ezihlobene eduze. Lokhu kutholakala kungasiza ekucaciseni imvelaphi kanye nokuvela kwe-ribosome ye-mitochondrial, eyaziwa nge-rRNA yayo encishisiwe kakhulu kanye nokuguquguquka okungavamile kokwakheka kwamaprotheni kuzo zonke izinhlobo.
Sabe sesiqhathanisa iphrotheni ye-msL2 nephrotheni ye-msL1 echazwe ngaphambilini, okuwukuphela kwephrotheni ye-ribosomal ye-microsporidia-specific etholakala ku-V. necatrix ribosome. Sasifuna ukuhlola ukuthi i-msL1 ne-msL2 zihlobene ngokuziphendukela kwemvelo yini. Ukuhlaziywa kwethu kubonise ukuthi i-msL1 ne-msL2 zihlala emgodini ofanayo esakhiweni se-ribosomal, kodwa zinezakhiwo ezihlukene eziyinhloko neziphezulu, okubonisa imvelaphi yazo ezizimele yokuziphendukela kwemvelo (Isithombe 6). Ngakho-ke, ukutholakala kwethu kwe-msL2 kunikeza ubufakazi bokuthi amaqembu ezinhlobo ze-eukaryotic ezihlangene angaguqula ngokuzimela amaprotheni e-ribosomal ahlukene ngokwesakhiwo ukuze akhokhele ukulahlekelwa yizingcezu ze-rRNA. Lokhu okutholakele kuyaphawuleka ngoba ama-ribosome amaningi e-cytoplasmic eukaryotic aqukethe iphrotheni engaguquki, kufaka phakathi umndeni ofanayo wamaprotheni e-ribosomal angu-81. Ukuvela kwe-msL1 ne-msL2 ezinhlotsheni ezahlukene ze-microsporidia ekuphenduleni ukulahlekelwa kwezingxenye ze-rRNA ezinde kusikisela ukuthi ukuwohloka kokwakheka kwama-molecule e-parasite kubangela ukuthi ama-parasite afune izinguquko ezihlawulelayo, okungagcina kuholele ekutholakaleni kwawo ezinhlakeni ezahlukene zama-parasite.
Ekugcineni, lapho imodeli yethu isiqediwe, saqhathanisa ukwakheka kwe-ribosome ye-E. cuniculi nalokho okwabikezelwa kusukela ochungechungeni lwe-genome. Amaprotheni amaningana e-ribosomal, afaka phakathi i-eL14, i-eL38, i-eL41, kanye ne-eS30, ngaphambili kwakucatshangwa ukuthi awekho ku-genome ye-E. cuniculi ngenxa yokungabikho okusobala kwama-homologues awo ku-genome ye-E. cuniculi. Ukulahlekelwa kwamaprotheni amaningi e-ribosomal nakho kubikezelwa kwamanye ama-parasite angaphakathi kweseli ancishisiwe kakhulu kanye nama-endosymbionts. Isibonelo, yize iningi lamagciwane aphilayo mahhala liqukethe umndeni ofanayo wamaprotheni e-ribosomal angu-54, angu-11 kuphela ale mindeni yamaprotheni ane-homologues etholakala ku-genome ngayinye ehlaziyiwe yamagciwane avinjelwe umninikhaya. Ukusekela lo mbono, ukulahleka kwamaprotheni e-ribosomal kuye kwabonwa ngokuhlola ku-V. necatrix kanye ne-P. locustae microsporidia, angenayo i-eL38 kanye ne-eL4131,32 amaprotheni.
Kodwa-ke, izakhiwo zethu zibonisa ukuthi yi-eL38, eL41, kanye ne-eS30 kuphela ezilahlekile ku-ribosome ye-E. cuniculi. Iphrotheni ye-eL14 yalondolozwa futhi isakhiwo sethu sabonisa ukuthi kungani le phrotheni ingatholakali ekusesheni kwe-homology (Isithombe 7). Ku-ribosomes ye-E. cuniculi, iningi lendawo yokubopha ye-eL14 ilahlekile ngenxa yokuwohloka kwe-ES39L ekhulisiwe yi-rRNA. Uma kungekho i-ES39L, i-eL14 ilahlekelwe yisakhiwo sayo sesibili, futhi yi-18% kuphela yochungechunge lwe-eL14 olwalufana ku-E. cuniculi kanye ne-S. cerevisiae. Lokhu kulondolozwa kochungechunge olubi kuyamangalisa ngoba ngisho ne-Saccharomyces cerevisiae kanye ne-Homo sapiens—izinto eziphilayo ezashintsha iminyaka eyizigidigidi eziyi-1.5—zihlanganyela ngaphezu kwama-51% ezinsalela ezifanayo ku-eL14. Lokhu kulahlekelwa okungavamile kokulondolozwa kuchaza ukuthi kungani i-E. cuniculi eL14 okwamanje ichazwa njengeprotheni ye-M970_061160 ecatshangelwayo hhayi njengeprotheni ye-ribosomal ye-eL1427.
kanye ne-Microsporidia ribosome ilahlekelwe yi-ES39L rRNA extension, eyasusa ingxenye yesiza sokubopha i-eL14 ribosomal protein. Uma kungekho i-ES39L, i-eL14 microspore protein ilahlekelwa yisakhiwo sesibili, lapho i-α-helix yangaphambili ebopha i-rRNA ibola ibe yi-loop encane yobude. b Ukuhlelwa kwe-multiple sequence kukhombisa ukuthi i-eL14 protein ilondolozwe kakhulu ezinhlotsheni ze-eukaryotic (ubunikazi be-sequence obungu-57% phakathi kwemvubelo nama-homologue abantu), kodwa ilondolozwe kabi futhi ihlukane kuma-microsporidia (lapho kungekho ngaphezu kwama-24% ezinsalela ezifanayo ne-eL14 homologue). kusuka ku-S. cerevisiae noma i-H. sapiens). Lokhu kulondolozwa kwe-sequence okungekuhle kanye nokwehluka kwesakhiwo sesibili kuchaza ukuthi kungani i-eL14 homologue ingakaze itholakale ku-E. cuniculi nokuthi kungani le protein kucatshangwa ukuthi ilahlekile ku-E. cuniculi. Ngokuphambene nalokho, i-E. cuniculi eL14 yayichazwe ngaphambilini njengeprotheni ye-M970_061160 ecatshangelwayo. Lokhu kuqaphela kusikisela ukuthi ukuhlukahluka kwe-genome ye-microsporidia okwamanje kulinganiselwa ngokweqile: ezinye izakhi zofuzo okucatshangwa ukuthi zilahlekile ku-microsporidia empeleni zilondolozwe, yize zisezinhlotsheni ezihlukene kakhulu; esikhundleni salokho, ezinye kucatshangwa ukuthi zibhala izakhi zofuzo ze-microsporidia zamaprotheni athile ezibungu (isb., iphrotheni ecatshangelwayo i-M970_061160) empeleni zibhala amaprotheni ahlukahlukene kakhulu atholakala kwamanye ama-eukaryotes.
Lokhu okutholakele kusikisela ukuthi ukuguquka kwe-rRNA kungaholela ekulahlekelweni okukhulu kokulondolozwa kokulandelana kwamaprotheni e-ribosomal aseduze, okwenza la maprotheni angatholakali ekusesheni kwe-homology. Ngakho-ke, singase silinganisele izinga langempela lokuwohloka kwamangqamuzana ezintweni eziphilayo ezincane ze-genome, njengoba amanye amaprotheni okucatshangwa ukuthi alahlekile empeleni ayaqhubeka, yize esesimweni esishintshile kakhulu.
Ama-parasite angawugcina kanjani umsebenzi wemishini yawo yama-molecule ngaphansi kwezimo zokuncipha okukhulu kwe-genome? Ucwaningo lwethu luphendula lo mbuzo ngokuchaza isakhiwo esiyinkimbinkimbi sama-molecule (i-ribosome) se-E. cuniculi, into ephilayo ene-genome encane kakhulu ye-eukaryotic.
Sekuyiminyaka ecishe ibe ngamashumi amabili kwaziwa ukuthi ama-molecule e-protein kanye ne-RNA kuma-microbial parasites avame ukuhluka kuma-molecule awo afanayo ezinhlotsheni eziphilayo ezikhululekile ngoba awanazo izikhungo zokulawula ikhwalithi, ancishiswa abe ngu-50% wobukhulu bawo kuma-free-living microbes, njll. izinguquko eziningi eziwohlozayo eziphazamisa ukugoqeka nokusebenza. Isibonelo, ama-ribosome ezinto eziphilayo ezincane ze-genome, okuhlanganisa ama-parasites amaningi angaphakathi kweseli kanye nama-endosymbionts, kulindeleke ukuthi antule amaprotheni amaningana e-ribosomal kanye nengxenye eyodwa kwezintathu zama-nucleotide e-rRNA uma kuqhathaniswa nezinhlobo eziphilayo ezikhululekile 27, 29, 30, 49. Kodwa-ke, indlela la ma-molecule asebenza ngayo kuma-parasites isalokhu iyimfihlakalo, ifundwa kakhulu ngokusebenzisa ama-genomics aqhathaniswayo.
Ucwaningo lwethu lubonisa ukuthi isakhiwo sama-macromolecule singaveza izici eziningi zokuziphendukela kwemvelo okunzima ukuzikhipha ezifundweni zendabuko zokuqhathanisa izakhi zofuzo zama-parasite angaphakathi kwamangqamuzana kanye nezinye izinto eziphilayo ezivinjelwe umninikhaya (Isithombe Esingeziwe 7). Isibonelo, isibonelo seprotheyini ye-eL14 sibonisa ukuthi singalinganisa izinga langempela lokuwohloka kwe-molecule yedivayisi ezinhlotsheni zama-parasite. Ama-parasite e-encephalitic manje kukholelwa ukuthi anamakhulu ama-gene aqondene nama-microsporidia. Kodwa-ke, imiphumela yethu ikhombisa ukuthi ezinye zalezi zakhi zofuzo ezibonakala ziqondile empeleni ziyizinhlobo ezihlukile kakhulu zama-gene ezivamile kwamanye ama-eukaryotes. Ngaphezu kwalokho, isibonelo seprotheyini ye-msL2 sibonisa ukuthi singazinaki kanjani amaprotheni amasha e-ribosomal futhi singazinaki okuqukethwe yimishini yama-molecule yama-parasite. Isibonelo sama-molecule amancane sibonisa ukuthi singazinaki kanjani izinto ezintsha ezihlakaniphile ezakhiweni zama-molecule ama-parasite ezingabanika umsebenzi omusha webhayoloji.
Uma zihlanganiswa, le miphumela ithuthukisa ukuqonda kwethu umehluko phakathi kwezakhiwo zama-molecule zezinto eziphilayo ezivinjelwe umninikhaya kanye nezinye eziphilayo eziphilayo ezikhululekile. Sibonisa ukuthi imishini yama-molecule, okucatshangwa ukuthi incishisiwe isikhathi eside, iyawohloka, futhi ingaphansi kwezinguquko ezahlukahlukene eziwohlozayo, esikhundleni salokho ineqoqo lezici zesakhiwo ezingavamile ezinganakwa ngokuhlelekile.
Ngakolunye uhlangothi, izingcezu ze-rRNA ezingezona ezinkulu kanye nezingcezu ezihlanganisiwe esizithole kuma-ribosome e-E. cuniculi zisikisela ukuthi ukunciphisa i-genome kungashintsha ngisho nalezo zingxenye zemishini eyisisekelo yama-molecule ezigcinwe ezizindeni ezintathu zokuphila - ngemva kweminyaka ecishe ibe yizigidi eziyizinkulungwane ezingu-3.5. ukuvela okuzimele kwezinhlobo.
Izingcezu ze-rRNA ezingenamaqhubu nezihlanganisiwe kuma-ribosome e-E. cuniculi zithakazelisa kakhulu ekukhanyeni kwezifundo zangaphambilini zama-molecule e-RNA kuma-bacteria e-endosymbiotic. Isibonelo, kuma-aphid endosymbiont Buchnera aphidicola, ama-molecule e-rRNA kanye ne-tRNA aboniswe enezakhiwo ezizwela izinga lokushisa ngenxa yokuthambekela kokwakheka kwe-A+T kanye nengxenye ephezulu yama-base pairs angewona ama-canonical20,50. Lezi zinguquko ku-RNA, kanye nezinguquko kuma-molecule e-protein, manje kucatshangwa ukuthi zibangela ukuthembela ngokweqile kwama-endosymbiont kubalingani kanye nokungakwazi kwama-endosymbiont ukudlulisa ukushisa 21, 23. Nakuba i-microsporidia rRNA enama-parasitic inezinguquko ezihlukile ngokwesakhiwo, uhlobo lwalezi zinguquko lubonisa ukuthi ukuzinza kokushisa okunciphile kanye nokuthembela okuphezulu kuma-protein e-chaperone kungaba yizici ezivamile zama-molecule e-RNA ezintweni eziphilayo ezinama-genome ancishisiwe.
Ngakolunye uhlangothi, izakhiwo zethu zibonisa ukuthi i-parasite microsporidia ithuthukise ikhono eliyingqayizivele lokumelana nezingcezu ze-rRNA kanye namaprotheni ezigcinwe kabanzi, ithuthukisa ikhono lokusebenzisa ama-metabolites amancane amaningi futhi atholakala kalula njengezinto ezilingiswayo zesakhiwo se-rRNA ewohlokile kanye nezingcezu zamaprotheni. Ukuwohloka kwesakhiwo sama-molecule. . Lo mbono usekelwa iqiniso lokuthi ama-molecule amancane akhokhela ukulahlekelwa kwezingcezu zamaprotheni ku-rRNA kanye nama-ribosome e-E. cuniculi abopha ezinsaleleni ezithile ze-microsporidia kumaprotheni e-uL15 kanye ne-eL30. Lokhu kusikisela ukuthi ukubopha kwama-molecule amancane kuma-ribosome kungaba umkhiqizo wokukhetha okuhle, lapho ukuguqulwa kwe-Microsporidia okuqondile kumaprotheni e-ribosomal kukhethwe khona ngenxa yamandla awo okukhulisa ukusondelana kwama-ribosome kuma-molecule amancane, okungaholela ezintweni eziphilayo ze-ribosomal ezisebenza kahle kakhulu. Lokhu kutholwa kwembula ubuhlakani obuhlakaniphile esakhiweni sama-molecule sama-microbial parasites futhi kusinika ukuqonda okungcono kokuthi izakhiwo zama-molecule ama-parasite zigcina kanjani umsebenzi wazo naphezu kokuvela okunciphisayo.
Okwamanje, ukuhlonza lawa ma-molecule amancane akukacaci. Akukacaci ukuthi kungani ukubonakala kwalawa ma-molecule amancane esakhiweni se-ribosomal kuhluka phakathi kwezinhlobo ze-microsporidia. Ikakhulukazi, akucaci ukuthi kungani ukubopha kwe-nucleotide kubonakala kuma-ribosome e-E. cuniculi kanye ne-P. locustae, hhayi kuma-ribosome e-V. necatrix, naphezu kokuba khona kwe-F170 residue kumaprotheni e-eL20 kanye ne-K172 e-V. necatrix. Lokhu kususwa kungabangelwa yi-residue 43 uL6 (etholakala eduze kwesikhwama sokubopha i-nucleotide), okuyi-tyrosine ku-V. necatrix hhayi i-threonine ku-E. cuniculi kanye ne-P. locustae. Uchungechunge olunephunga elimnandi lwe-Tyr43 lungaphazamisa ukubopha kwe-nucleotide ngenxa yokuhlangana kwe-steric. Ngaphandle kwalokho, ukususwa kwe-nucleotide okubonakalayo kungabangelwa yisisombululo esiphansi se-cryo-EM imaging, okuvimbela ukumodelwa kwezingcezu ze-ribosomal ze-V. necatrix.
Ngakolunye uhlangothi, umsebenzi wethu uphakamisa ukuthi inqubo yokubola kwe-genome ingaba amandla okusungula izinto. Ikakhulukazi, isakhiwo se-E. cuniculi ribosome sibonisa ukuthi ukulahleka kwezingcezu ze-rRNA kanye namaprotheni ku-microsporidia ribosome kudala ingcindezi yokuziphendukela kwemvelo ekhuthaza izinguquko esakhiweni se-ribosome. Lezi zinhlobo zenzeka kude nendawo esebenzayo ye-ribosome futhi zibonakala zisiza ekugcineni (noma ukubuyisela) ukuhlangana kwe-ribosome okuhle obekungaphazanyiswa yi-rRNA encishisiwe. Lokhu kusikisela ukuthi ukusungula okusha okukhulu kwe-microsporidia ribosome kubonakala sengathi kuguquke kwaba nesidingo sokuvimbela ukukhukhuleka kwezakhi zofuzo.
Mhlawumbe lokhu kuboniswa kangcono ukubopha kwe-nucleotide, okungakaze kubonwe kwezinye izinto eziphilayo kuze kube manje. Iqiniso lokuthi izinsalela zokubopha i-nucleotide zikhona kuma-microsporidia ajwayelekile, kodwa hhayi kwamanye ama-eukaryotes, lisikisela ukuthi izindawo zokubopha i-nucleotide azizona nje izinsalela ezilindele ukunyamalala, noma indawo yokugcina yokuthi i-rRNA ibuyiselwe esimweni sama-nucleotide ngamanye. Esikhundleni salokho, le ndawo ibonakala njengesici esiwusizo esingaba savela emizuliswaneni eminingana yokukhetha okuhle. Izindawo zokubopha i-nucleotide zingaba umkhiqizo ohlukile wokukhetha kwemvelo: uma i-ES39L yonakele, ama-microsporidia aphoqeleka ukufuna isinxephezelo sokubuyisela i-ribosome biogenesis efanele uma kungekho i-ES39L. Njengoba le nucleotide ingalingisa ukuxhumana kwama-molecule kwe-A3186 nucleotide ku-ES39L, i-nucleotide molecule iba yisisekelo se-ribosome, ukubopha kwayo okuthuthukiswa kakhulu ngokuguqulwa kochungechunge lwe-eL30.
Ngokuphathelene nokuvela kwama-molecule ezilokazane zangaphakathi kwamangqamuzana, ucwaningo lwethu lubonisa ukuthi amandla okukhethwa kwemvelo kukaDarwin kanye nokuvela kwama-genetic kokubola kwe-genome azisebenzi ngesikhathi esifanayo, kodwa kuyashintshashintsha. Okokuqala, ukuvela kwama-genetic kususa izici ezibalulekile zama-biomolecule, okwenza isinxephezelo sidingeke kakhulu. Kuphela lapho izilokazane zanelisa lesi sidingo ngokukhethwa kwemvelo kukaDarwin lapho ama-macromolecule azo ezoba nethuba lokuthuthukisa izici zawo ezihlaba umxhwele nezintsha kakhulu. Okubalulekile, ukuvela kwezindawo zokubopha i-nucleotide ku-E. cuniculi ribosome kusikisela ukuthi le ndlela yokulahlekelwa yinzuzo yokuvela kwama-molecule ayigcini nje ngokunciphisa izinguquko ezilimazayo, kodwa ngezinye izikhathi inikeza imisebenzi emisha ngokuphelele kuma-macromolecule ezilokazane.
Lo mbono uhambisana nombono kaSewell Wright wokulingana okuhambayo, othi uhlelo oluqinile lokukhetha kwemvelo lukhawulela ikhono lezinto eziphilayo lokuqamba izinto ezintsha51,52,53. Kodwa-ke, uma ukukhukhuleka kwezakhi zofuzo kuphazamisa ukukhetha kwemvelo, lokhu kukhukhuleka kungadala izinguquko ezingezona ngokwazo eziguquguqukayo (noma ezilimazayo) kodwa eziholela ezinguqukweni ezengeziwe ezihlinzeka ngokuqina okuphezulu noma umsebenzi omusha wezinto eziphilayo. Uhlaka lwethu lusekela lo mbono ngokubonisa ukuthi uhlobo olufanayo lokuguquka olunciphisa ukugoqeka kanye nomsebenzi we-biomolecule lubonakala luyimbangela eyinhloko yokuthuthuka kwayo. Ngokuhambisana nemodeli yokuziphendukela kwemvelo ephumelela kuwo wonke umuntu, ucwaningo lwethu lubonisa ukuthi ukubola kwe-genome, okuvame ukubhekwa njengenqubo yokuwohloka, nakho kuyimbangela enkulu yokusungula izinto ezintsha, ngezinye izikhathi futhi mhlawumbe kaningi okuvumela ama-macromolecule ukuthi athole imisebenzi emisha ye-parasitic.