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Ukuvela kwezifunxi-gazi zibandakanya ukuchasana phakathi kokhetho lwendalo, olubangela ukuba izifunxi-gazi ziphucuke, kunye nemfuzo, ebangela ukuba izifunxi-gazi ziphulukane nemizila yemfuza kwaye ziqokelele iinguqulelo ezisusayo.Apha, ukuze uqonde indlela oku kuphikisana okwenzeka ngayo kwisikali se-macromolecule enye, sichaza isakhiwo se-cryo-EM se-ribosome ye-Encephalitozoon cuniculi, i-eukaryotic organism enye ye-genomes encinci kwindalo.Ukuncitshiswa okugqithisileyo kwe-rRNA kwi-E. cuniculi ribosomes kukhatshwa lutshintsho lwesakhiwo olungazange lubonwe ngaphambili, olufana nokuvela kwezikhonkco ze-rRNA ezazingaziwa ngaphambili kunye ne-rRNA ngaphandle kwamaqhuma.Ukongeza, i-E. cuniculi ribosome yasinda ekulahlekeni kwamaqhekeza kunye neeproteni ze-rRNA ngokuphuhlisa isakhono sokusebenzisa iimolekyuli ezincinci njengemilinganiso yesakhiwo samaqhekeza e-rRNA kunye neeproteni.Ngokubanzi, sibonisa ukuba izakhiwo ze-molecular ezicinga ukuba ziyancitshiswa, ziyancipha, kwaye zixhomekeke kwiinguqu eziphazamisayo zinenani leendlela zokubuyisela ezizigcina zisebenza nangona i-contraction ye-molecular iyancipha.
Ngenxa yokuba uninzi lwamaqela e-microbial parasites anezixhobo ezizodwa zemolekyuli zokuxhaphaza ababuki zindwendwe bazo, kuye kufuneke siphuhlise iindlela zonyango ezahlukeneyo kumaqela ahlukeneyo eentsholongwane1,2.Nangona kunjalo, ubungqina obutsha bubonisa ukuba ezinye iinkalo ze-parasite evolution ziyaguquguquka kwaye ziqikeleleka kakhulu, zibonisa isiseko esinokubakho songenelelo olubanzi lonyango kwi-microbial parasites3,4,5,6,7,8,9.
Umsebenzi wangaphambili uchonge umkhwa oqhelekileyo wokuziphendukela kwemvelo kwiintsholongwane ezibizwa ngokuba yi-genome reduction okanye i-genome decay10,11,12,13.Uphando lwangoku lubonisa ukuba xa ii-microorganisms zinikezela ubomi bazo obukhululekile kwaye zibe yi-intracellular parasites (okanye i-endosymbionts), i-genomes yazo ihamba ngokukhawuleza kodwa i-metamorphoses emangalisayo kwizigidi zeminyaka9,11.Kwinkqubo eyaziwa ngokuba yi-genome decay, ii-microbial parasites ziqokelela ukuguqulwa kwezinto eziguqukayo eziguqula izakhi zofuzo ezininzi ezibalulekileyo ngaphambili kwi-pseudogenes, ekhokelela ekulahlekeni kwemfuza ngokuthe ngcembe kunye nokuwa kwe-mutation14,15.Oku kudilika kunokutshabalalisa ukuya kuthi ga kwi-95% yemfuza kwezona zinto zindala ze-intracellular organisms xa kuthelekiswa neentlobo ezisondeleleneyo eziphilayo ezikhululekileyo.Ngaloo ndlela, i-evolution ye-intracellular parasites yi-tug-of-war phakathi kwamandla amabini aphikisanayo: ukhetho lwendalo lwe-Darwin, olukhokelela ekuphuculeni ama-parasites, kunye nokuwa kwe-genome, ukuphosa i-parasites ekulibaleni.Indlela i-parasite ekwazi ngayo ukuvela kule mpi yokutsalwa kwaye igcine umsebenzi wesakhiwo sayo se-molekyuli akukacaci.
Nangona indlela yokubola kwe-genome ingaqondwa ngokupheleleyo, ibonakala isenzeka ikakhulu ngenxa yokuphazamiseka rhoqo kwemfuzo.Ngenxa yokuba ii-parasites zihlala kwiindawo ezincinci, i-asexual, kunye ne-genetically limited populations, azikwazi ukuphelisa ngokufanelekileyo ukuguqulwa kokuguquka okwenzeka ngamanye amaxesha ngexesha lokuphindaphinda kwe-DNA.Oku kukhokelela ekuqokeleleni okungenakujikwa kweenguqu ezinobungozi kunye nokunciphisa i-parasite genome.Ngenxa yoko, i-parasite ayilahleki kuphela izakhi zofuzo ezingaseyomfuneko ukuba ziphile kwindawo ye-intracellular.Kukungakwazi kwabemi be-parasite ukuphelisa ngempumelelo utshintsho oluqhelekileyo olubangela ukuba olu tshintsho luqokelele kuyo yonke i-genome, kuquka neyona mizila ibalulekileyo.
Uninzi lokuqonda kwethu kwangoku ngokuncitshiswa kwe-genome kusekelwe kuphela ekuthelekisweni kolandelelwano lwe-genome, kunye nengqalelo encinci kwiinguqu ze-athomu zangempela ezenza imisebenzi yokugcina indlu kwaye zisebenze njengezinto ekujoliswe kuzo zamachiza.Uphononongo oluthelekisayo lubonise ukuba umthwalo we-demorious intracellular microbial mutations ubonakala ubeka phambili iiprotheyini kunye ne-nucleic acids ukuba zingahambi kakuhle kwaye zidibanise, zibenze zixhomekeke ngakumbi kwi-chaperone kunye ne-hypersensitive kwi-heat19,20,21,22,23.Ukongeza, ii-parasites ezahlukeneyo-ukuziphendukela kwemvelo okuzimeleyo ngamanye amaxesha zahlulwa malunga ne-2.5 yezigidigidi zeminyaka-zifumene ilahleko efanayo yamaziko okulawula umgangatho kwiiprotheyini zabo ze-protein5,6 kunye neendlela zokulungisa i-DNA24.Nangona kunjalo, kuncinci okwaziwayo malunga nefuthe lendlela yokuphila ye-intracellular kuzo zonke ezinye iipropathi ze-macromolecules zeselula, kubandakanya ukulungelelaniswa kwemolekyuli kumthwalo owandayo wotshintsho olucimayo.
Kulo msebenzi, ukuze siqonde ngcono ukuvela kweeprotheni kunye ne-nucleic acid ye-intracellular microorganisms, sinqume isakhiwo se-ribosomes ye-intracellular parasite Encephalitozoon cuniculi.I-E. cuniculi yintsholongwane efana ne-fungus yeqela le-parasitic microsporidia ene-eukaryotic genomes encinci ngokungaqhelekanga kwaye isetyenziswa njengezinto eziphilayo ezingumzekelo wokufunda ukubola kwe-genome25,26,27,28,29,30.Kungekudala, i-cryo-EM isakhiwo se-ribosome sinqunywe ngokunciphisa ngokulinganayo i-genomes ye-Microsporidia, i-Paranosema locustae, kunye ne-Vairimorpha necatrix31,32 (~ 3.2 Mb genome).Ezi zakhiwo zibonisa ukuba ilahleko ethile ye-rRNA yokukhulisa imbuyekezo ngokuphuhliswa koqhagamshelwano olutsha phakathi kweeprotheni ze-ribosomal ezingabamelwane okanye ukufumana iiprotheni ze-ribosomal ze-msL131,32 ezintsha.Iintlobo ze-Encephalitozoon (i-genome ~ 2.5 yezigidi ze-bp), kunye nesihlobo sabo esisondeleyo se-Ordospora, babonisa iqondo lokugqibela lokunciphisa i-genome kwi-eukaryotes - banegazi elingaphantsi kwe-2000 ye-protein-coding genes, kwaye kulindeleke ukuba i-ribosomes yazo ayinazo kuphela i-rRNA i-ribosome fragments (i-rRNA i-ribosome fragments) iiprotheni ze-ribosomal ngenxa yokunqongophala kwee-homologues kwi-E. cuniculi genome26,27,28.Ngoko ke, siye sagqiba kwelokuba i-E. cuniculi ribosome inokutyhila iindlela ezazingaziwa ngaphambili zokuziqhelanisa nemolekyuli ukubola kwegenome.
Ulwakhiwo lwethu lwe-cryo-EM lumele eyona ribosome incinci ye-eukaryotic cytoplasmic ukuba ibonakaliswe kwaye ibonelele malunga nokuba inqanaba lokugqibela lokuncitshiswa kwe-genome lichaphazela njani ulwakhiwo, ukudibanisa, kunye nokuvela komatshini wemolekyuli obalulekileyo kwiseli.Sifumanise ukuba i-E. cuniculi ribosome iphula uninzi lwemigaqo egcinwe ngokubanzi ye-RNA yokusonga kunye nendibano ye-ribosome, kwaye yafumanisa iprotein entsha ye-ribosomal eyayingaziwa ngaphambili.Ngokungalindelekanga, sibonisa ukuba i-microsporidia ribosomes iye yavelisa amandla okubopha iimolekyuli ezincinci, kwaye iqikelelwa ukuba ukunqunyulwa kwe-rRNA kunye neeproteni kuvusa inguqulelo yendalo enokuthi ekugqibeleni inike iimpawu eziluncedo kwi-ribosome.
Ukuphucula ukuqonda kwethu ukuguquka kweeprotheni kunye ne-nucleic acids kwi-intracellular organisms, sagqiba ekubeni sihlukanise i-E. cuniculi spores kwiinkcubeko zeeseli ezithintekayo ze-mammalian ukuze zihlambulule i-ribosomes yazo kwaye zinqume isakhiwo sezi ribosomes.Kunzima ukufumana inani elikhulu le-microsporidia ye-parasitic kuba i-microsporidia ayikwazi ukukhuliswa kwindawo yezondlo.Kunoko, ziyakhula zize zivelise kuphela ngaphakathi kwiseli yomkhosi.Ngoko ke, ukufumana i-E. cuniculi biomass yokucoca i-ribosome, sosulela umgca weseli yezintso ezincancisayo i-RK13 nge-E. cuniculi spores kwaye sikhulise ezi seli zosulelekileyo iiveki ezininzi ukuvumela i-E. cuniculi ukuba ikhule kwaye iphindaphindeke.Ukusebenzisa i-cell monolayer echaphazelekayo malunga nesiqingatha semitha yesikwere, sakwazi ukucoca malunga ne-300 mg ye-Microsporidia spores kwaye siyisebenzise ukwahlula i-ribosomes.Emva koko saphazamisa i-spores ehlambulukileyo ngamaso eglasi kwaye sahlukanisa i-ribosomes ekrwada sisebenzisa i-stepwise polyethylene glycol fractionation ye-lysates.Oku kusivumele ukuba sifumane malunga nama-300 µg ekrwada ye-E. cuniculi ribosomes kuhlalutyo lwesakhiwo.
Emva koko siqokelele imifanekiso ye-cryo-EM sisebenzisa iisampulu ze-ribosome ezifunyenweyo kwaye senza le mifanekiso sisebenzisa iimaski ezihambelana ne-ribosomal subunit enkulu, i-subunit encinci, kunye ne-subunit encinci.Ngethuba le nkqubo, siqokelele imifanekiso malunga ne-108,000 ye-ribosomal particles kunye ne-computing cryo-EM imifanekiso kunye nesisombululo se-2.7 Å (Amanani abongezelelweyo 1-3).Emva koko sasebenzisa imifanekiso ye-cryoEM kwimodeli ye-rRNA, iprotheni ye-ribosomal, kunye ne-hibernation factor Mdf1 ehambelana ne-E. cuniculi ribosomes (Umfanekiso 1a, b).
a Isakhiwo se-E. cuniculi ribosome entsonkothileyo ene-hibernation factor Mdf1 (pdb id 7QEP).b Imephu ye-hibernation factor Mdf1 ehambelana ne-E. cuniculi ribosome.c Imephu yolwakhiwo lwesibini ithelekisa i-rRNA efunyenweyo kwiintlobo zeMicrosporidian kunye nezakhiwo ezaziwayo zeribosomal.Iiphaneli zibonisa indawo ye-rRNA fragments (ES) kunye neendawo ezisebenzayo ze-ribosome, kubandakanywa nesayithi ye-decoding (DC), i-sarcinicin loop (SRL), kunye ne-peptidyl transferase center (PTC).d Ubuninzi be-electron obuhambelana ne-peptidyl transferase center ye-E. cuniculi ribosome icebisa ukuba le ndawo ye-catalytic inesakhiwo esifanayo kwi-E. cuniculi parasite kunye nemikhosi yayo, kuquka i-H. sapiens.e, f Uxinaniso lwe-electron oluhambelanayo lweziko le-decoding (e) kunye nolwakhiwo lweskim seziko lokuchaza (f) lubonisa ukuba i-E. cuniculi ineentsalela ze-U1491 endaweni ye-A1491 (inombolo ye-E. coli) kwezinye ii-eukaryotes ezininzi.Olu tshintsho lucebisa ukuba i-E. cuniculi inokuba novakalelo kwii-antibiotics ezijolise kule ndawo isebenzayo.
Ngokuchasene nezakhiwo ezisele zisekwe ngaphambili ze-V. necatrix kunye ne-P. locustae ribosomes (zombini izakhiwo zimele i-microsporidia usapho lwe-Nosematidae kwaye zifana kakhulu enye kwenye), i-31,32 E. cuniculi ribosomes ingena kwiinkqubo ezininzi ze-rRNA kunye nokuqhekeka kweprotheni.I-denaturation eyongezelelweyo (Amanani abongezelelweyo 4-6).Kwi-rRNA, olona tshintsho lubalaseleyo lubandakanya ukulahleka okupheleleyo kweqhekeza le-25S rRNA elikhulisiwe ES12L kunye nokuncipha kwenxalenye ye-h39, h41, kunye ne-H18 helices (umzobo 1c, umzobo we-Supplementary Fig. 4).Phakathi kweeprotheyini ze-ribosomal, olona tshintsho lubalaseleyo lubandakanya ilahleko epheleleyo yeprotheyini ye-eS30 kunye nokuncipha kwe-eL8, eL13, eL18, eL22, eL29, eL40, uS3, uS9, uS14, uS17, kunye neeprotheni ze-eS7 (Amanani abongezelelweyo 4, 5).
Ngoko ke, ukuncitshiswa okugqithisileyo kweegenomes zeentlobo ze-Encephalotozoon/Ordospora kubonakaliswe kulwakhiwo lwe-ribosome: I-E. cuniculi ribosomes ifumana eyona lahleko imangalisayo yesiqulatho seprotheyini kwi-eukaryotic cytoplasmic ribosomes exhomekeke kuphawu lwesakhiwo, kwaye ayinazo ezo rRNA kunye namaqhekeza eprotein akwidomeyini ebanzi ye-inservukas kuphela, kodwa ikwayiyo imimandla ebanzi yeprotein.Ulwakhiwo lwe-E. cuniculi ribosome lubonelela ngemodeli yokuqala yeemolekyuli zolu tshintsho kwaye lutyhila iziganeko zokuziphendukela kwemvelo eziye zangahoywa zizo zombini i-genomics yokuthelekisa kunye nezifundo ze-intracellular biomolecular structure (I-Supplementary Fig. 7).Apha ngezantsi, sichaza nganye yezi ziganeko kunye nemvelaphi yazo enokubakho yendaleko kunye nefuthe lazo elinokubakho kumsebenzi weribosome.
Siye safumanisa ukuba, ukongeza kwi-rRNA truncations ezinkulu, i-E. cuniculi ribosomes inomahluko we-rRNA kwenye yeziza ezisebenzayo.Nangona i-peptidyl transferase center ye-E. cuniculi ribosome inesakhiwo esifanayo njengenye i-eukaryotic ribosomes (Umfanekiso we-1d), i-decoding center iyahluka ngenxa yokuhlukahluka kolandelelwano kwi-nucleotide 1491 (i-E. coli inombolo, i-Fig. 1e, f).Olu qwalaselo lubalulekile kuba indawo yokuchaza i-ribosomes ye-eukaryotic idla ngokuqulatha iintsalela ze-G1408 kunye ne-A1491 xa kuthelekiswa neentsalela zohlobo lwebhaktiriya i-A1408 kunye ne-G1491.Olu lwahluko luphantsi kobuntununtunu obahlukeneyo bebhaktiriya kunye ne-eukaryotic ribosomes kwintsapho ye-aminoglycoside ye-ribosomal antibiotics kunye nezinye iimolekyuli ezincinci ezijolise kwindawo yokucacisa.Kwindawo ye-decoding ye-E. cuniculi ribosome, intsalela ye-A1491 yathatyathelwa indawo nge-U1491, enokuthi idale ujongano olubophelelayo olulodwa lweeamolekyuli ezincinci ezijolise kule ndawo isebenzayo.Ukwahluka okufanayo kwe-A14901 kukho nakwezinye i-microsporidia ezifana ne-P. locustae kunye ne-V. necatrix, ebonisa ukuba ixhaphake phakathi kweentlobo ze-microsporidia (Umfanekiso 1f).
Ngenxa yokuba iisampulu zethu ze-E. cuniculi ribosome zazibekwe zodwa kwi-metabolically inactive spores, siye savavanya imephu ye-cryo-EM ye-E. cuniculi ngokubophelela kwi-ribosome echazwe ngaphambili phantsi koxinzelelo okanye iimeko zendlala.Imiba ye-Hibernation 31,32,36,37, 38. Sifanise isakhiwo esenziwe ngaphambili se-hibernating ribosome kunye nemephu ye-cryo-EM ye-E. cuniculi ribosome.Kwi-docking, i-S. cerevisiae ribosomes isetyenziswe kwi-complex kunye ne-hibernation factor Stm138, i-ribosomes yeenkumbi kwi-complex kunye ne-Lso232 factor, kunye ne-V. necatrix ribosomes eyinkimbinkimbi kunye ne-Mdf1 kunye ne-Mdf231.Kwangaxeshanye, sifumene ingxinano ye-cryo-EM ehambelana nenye into Mdf1.Ngokufana ne-Mdf1 ebophelela kwi-V. necatrix ribosome, i-Mdf1 iphinda ibophelele kwi-E. cuniculi ribosome, apho ivimba indawo ye-E ye-ribosome, mhlawumbi inceda ukwenza i-ribosomes ifumaneke xa i-parasite spores ingasebenzi kwi-metabolically inctivation (Figure 2).).
I-Mdf1 ivala indawo ye-E ye-ribosome, ebonakala inceda ukuvuselela i-ribosome xa i-parasite spores ingasebenzi.Kwisakhiwo se-E. cuniculi ribosome, sifumene ukuba i-Mdf1 yenza uqhagamshelwano olungaziwa ngaphambili kunye ne-L1 ribosome stem, inxalenye ye-ribosome eyenza ukukhululwa kwe-tRNA e-deacylated kwi-ribosome ngexesha le-protein synthesis.Aba bafowunelwa bacebisa ukuba i-Mdf1 iyahlukana ne-ribosome isebenzisa indlela efanayo ne-deacetylated tRNA, inika inkcazo enokwenzeka malunga nendlela i-ribosome esusa ngayo i-Mdf1 ukuze iphinde isebenze i-protein synthesis.
Nangona kunjalo, ulwakhiwo lwethu luveze unxibelelwano olungaziwayo phakathi kweMdf1 kunye nomlenze we-ribosome we-L1 (inxalenye ye-ribosome enceda ukukhulula i-tRNA e-deacylated kwi-ribosome ngexesha leprotein synthesis).Ngokukodwa, i-Mdf1 isebenzisa abafowunelwa abafanayo njengecandelo le-elbow ye-molecule ye-tRNA ye-deacylated (umzobo 2).Le modeli yemolekyuli eyayingaziwa ngaphambili ibonise ukuba i-Mdf1 iyahlukana ne-ribosome isebenzisa indlela efanayo ne-tRNA ye-deacetylated, echaza indlela i-ribosome esusa ngayo le nto ye-hibernation ukuze iphinde isebenze i-protein synthesis.
Xa sisakha imodeli ye-rRNA, safumanisa ukuba i-E. cuniculi ribosome ineziqhekeza ze-rRNA ezigoqwe ngokungaqhelekanga, esazibiza ngokuba yi-fused rRNA (Fig. 3).Kwii-ribosomes ezithatha imimandla emithathu yobomi, i-rRNA igoqa kwizakhiwo apho ezininzi iziseko ze-rRNA zinokuba zizibini ezisisiseko kunye nokugoqa kunye okanye zidibanise ne-ribosomal proteins38,39,40.Nangona kunjalo, kwi-E. cuniculi ribosomes, ii-rRNA zibonakala ziwaphula lo mgaqo usongwayo ngokuguqula ezinye zeeheliksi zazo zibe yimimandla ye-rRNA engatyhilekanga.
Isakhiwo se-H18 25S rRNA helix kwi-S. cerevisiae, V. necatrix, kunye ne-E. cuniculi.Ngokuqhelekileyo, kwii-ribosomes ezithatha imimandla emithathu yobomi, le khonkco idibanisa kwi-RNA helix equlethe i-24 ukuya kwi-34 intsalela.Kwi-Microsporidia, ngokuchaseneyo, le ngqungquthela ye-rRNA iyancitshiswa ngokuthe ngcembe ibe yi-uridine-stranded single-rich linkers equlethe iintsalela ze-12 kuphela.Uninzi lwezi ntsalela zichanabeke kwizinyibilikisi.Umzobo ubonisa ukuba i-microsporidia ye-parasitic ibonakala iphula imigaqo jikelele ye-rRNA ukusonga, apho iziseko ze-rRNA zihlala zidityaniswa kwezinye iziseko okanye zibandakanyeka kwi-rRNA-protein interactions.Kwi-microsporidia, ezinye iziqwenga ze-rRNA zithatha i-fold engathandekiyo, apho i-rRNA helix yangaphambili iba yi-fragment ene-stranded fragment eyandiswe phantse kumgca othe ngqo.Ubukho bale mimandla ingaqhelekanga ivumela i-microsporidia rRNA ukuba ibophe amaqhekeza e-rRNA ekude isebenzisa inani elincinci leziseko ze-RNA.
Umzekelo obalaseleyo wolu tshintsho lwenguquko unokubonwa kwi-H18 25S rRNA helix (umzobo 3).Kwiintlobo ezivela kwi-E. coli ukuya ebantwini, iziseko ze-rRNA helix ziqulethe i-24-32 nucleotides, eyenza i-helix engaqhelekanga.Kwizakhiwo ezichongiweyo ze-ribosomal zangaphambili ezivela kwi-V. necatrix kunye ne-P. locustae, i-31,32 iziseko ze-H18 helix zikhutshwe ngokuyinxenye, kodwa i-nucleotide base pairing igcinwe.Nangona kunjalo, kwi-E. cuniculi eliqhekeza le-rRNA liba lelona khonkco lifutshane 228UUUGU232 kunye ne-301UUUUUUUU307.Ngokungafaniyo namaqhekeza aqhelekileyo e-rRNA, ezi zikhonkco zityebileyo nge-uridine azikhohlisi okanye zinxibelelane kakhulu neeproteni ze-ribosomal.Endaweni yoko, bamkela i-solvent-evulekileyo kunye nezakhiwo ezivuleke ngokupheleleyo apho imicu ye-rRNA yandiswa phantse ngokuthe tye.Oku kuhambelana okunwetshiweyo kuchaza indlela u-E. cuniculi asebenzisa ngayo iziseko ze-RNA ezili-12 kuphela ukuzalisa i-33 Å gap phakathi kwe-H16 kunye ne-H18 rRNA helices, ngelixa ezinye iintlobo zifuna ubuncinane iziseko ze-rRNA ngokuphindwe kabini ukuvala isithuba.
Ke ngoko, sinokubonisa ukuba, ngokusonga ngamandla okungathandekiyo, i-parasitic microsporidia iphuhlise isicwangciso sokwenza ikhontrakthi naloo macandelo e-rRNA ahlala elondolozwe ngokubanzi kuzo zonke iintlobo zezilwanyana kwimimandla emithathu yobomi.Kuyabonakala ukuba, ngokuqokelela iinguqu eziguqula iihelikhwe ze-rRNA zibe zikhonkco ezimfutshane ze-poly-U, i-E. cuniculi inokwenza amaqhekeza e-rRNA angaqhelekanga aqulethe iinucleotide ezimbalwa kangangoko kunokwenzeka ukuze kuhlanganiswe amaqhekeza e-distal rRNA.Oku kunceda ukucacisa indlela i-microsporidia ephumelele ngayo ukunciphisa ngokumangalisayo kwisiseko sabo se-molecular structure ngaphandle kokulahlekelwa yintembeko yabo yesakhiwo kunye nokusebenza.
Enye into engaqhelekanga ye-E. cuniculi rRNA yimbonakalo ye-rRNA ngaphandle kokuqina (umzobo 4).Amaqhuqhuva ziinucleotides ezingenazo izibini ezisisiseko ezijijwa ngaphandle kweRNA helix endaweni yokuzifihla kuyo.Uninzi lweeprotrusions ze-rRNA zisebenza njengezinto zokuncamathelisa imolekyuli, zinceda ukubopha iiproteni ezikufutshane ne-ribosomal okanye ezinye iziqwenga ze-rRNA.Ezinye ze-bulges zisebenza njengeehenjisi, zivumela i-rRNA helix ukuba iguquke kwaye idibanise ngokufanelekileyo kwiprotheyini evelisa i-41.
i-An rRNA protrusion (S. cerevisiae numbering) ayikho kwi-E. cuniculi ribosome structure, kodwa ikhona kwezinye ii-eukaryotes b E. coli, S. cerevisiae, H. sapiens, kunye ne-E. cuniculi ribosomes zangaphakathi.izifunxi-gazi azinawo amaqhuqhuva e-rRNA akudala, alondolozwe kakhulu.Ezi ngqindilili zizinzisa isakhiwo se-ribosome;ngoko ke, ukungabikho kwabo kwi-microsporidia kubonisa ukuzinza okuncitshisiweyo kwe-rRNA ukugoqa kwii-microsporidia parasites.Ukuthelekisa kunye neziqu ze-P (i-L7 / L12 iziqu kwiibhaktheriya) zibonisa ukuba ukulahleka kwe-rRNA i-bumps ngamanye amaxesha ihambelana nokubonakala kwamatsha amatsha ecaleni kwe-bumps elahlekileyo.I-H42 helix kwi-23S / 28S rRNA ine-bulge yakudala (U1206 e-Saccharomyces cerevisiae) eqikelelwa ukuba ubuncinane i-3.5 yezigidigidi zeminyaka ubudala ngenxa yokukhusela kwayo kwimimandla emithathu yobomi.Kwi-microsporidia, le ngqungquthela iyapheliswa.Nangona kunjalo, i-bulge entsha yavela ecaleni kwe-bulge elahlekileyo (i-A1306 kwi-E. cuniculi).
Ngokumangalisayo, safumanisa ukuba i-E. cuniculi ribosomes ayinayo i-rRNA i-bulges efunyenwe kwezinye iintlobo, kubandakanywa ngaphezu kwe-30 i-bulges egcinwe kwezinye i-eukaryotes (umzobo 4a).Le lahleko iphelisa unxibelelwano oluninzi phakathi kwe-ribosomal subunits kunye ne-rRNA helices ekufutshane, ngamanye amaxesha idala i-voids enkulu egobileyo ngaphakathi kwe-ribosome, okwenza i-E. cuniculi ribosome ibe ne-porous ngakumbi xa kuthelekiswa ne-ribosomes yendabuko (Fig. 4b).Ngokucacileyo, sifumene ukuba uninzi lwala maqhuma nawo alahlekileyo kwizakhiwo ezichongiweyo ze-V. necatrix kunye ne-P. locustae ribosome, ezazingahoywa luhlalutyo lwangaphambili lwesakhiwo31,32.
Ngamanye amaxesha ukulahlekelwa kwe-rRNA i-bulges ihamba kunye nokuphuhliswa kwamatsha amatsha ecaleni kwe-bulge elahlekileyo.Ngokomzekelo, i-ribosomal P-stem iqulethe i-U1208 bulge (kwi-Saccharomyces cerevisiae) eyasinda kwi-E. coli ukuya ebantwini kwaye ngoko ke kuqikelelwa ukuba i-3.5 yeminyaka eyibhiliyoni ubudala.Ngexesha leprotein synthesis, le qhuma inceda i-P stem ukuba ihambe phakathi kwe-conformations evulekileyo kunye nevaliweyo ukuze i-ribosome ikwazi ukufumana izinto zokuguqulela kwaye ihanjiswe kwindawo esebenzayo.Kwi-E. cuniculi ribosomes, oku kungqimba akukho;nangona kunjalo, i-thickening entsha (i-G883) efumaneka kuphela kwizibini ezisisiseko ezintathu zinokufaka isandla ekubuyiseleni ukuguquguquka okufanelekileyo kwe-P stem (Umfanekiso 4c).
Idatha yethu kwi-rRNA ngaphandle kwe-bulges icebisa ukuba ukunciphisa i-rRNA akukhawulelwanga ekulahlekeni kwezinto ze-rRNA kumphezulu we-ribosome, kodwa kunokubandakanya i-ribosome nucleus, ukudala i-parasite-specific molecular defect engazange ichazwe kwiiseli eziphilayo zamahhala.iintlobo eziphilayo ziyajongwa.
Emva kokulinganisa iiprotheni ze-ribosomal ze-canonical kunye ne-rRNA, sifumene ukuba i-ribosomal components eziqhelekileyo azikwazi ukucacisa iinxalenye ezintathu zomfanekiso we-cryo-EM.Ezibini zala maqhekeza ziamolekyu ezincinci ngobukhulu (umzobo 5, umzobo owongezelelweyo 8).Icandelo lokuqala lixutywe phakathi kweeprotheni ze-ribosomal uL15 kunye ne-eL18 kwindawo edla ngokuhlala kwi-C-terminus ye-eL18, efinyeziweyo kwi-E. cuniculi.Nangona singenako ukufumanisa ukuba ngubani le molekyuli, ubungakanani kunye nokumila kwesi siqithi soxinaniso kuchazwa kakuhle kubukho beemolekyuli ze-spermidine.Ukubophelela kwayo kwi-ribosome kuzinziswa yi-microsporidia-specific mutations kwi-UL15 proteins (Asp51 kunye ne-Arg56), ebonakala ngathi inyusa ukudibanisa kwe-ribosome yale molekyuli encinci, njengoko ivumela i-UL15 ukuba idibanise i-molecule encinci kwisakhiwo se-ribosomal.Umzobo owongezelelweyo 2).8, idatha eyongezelelweyo 1, 2).
I-Cryo-EM imaging ebonisa ubukho be-nucleotides ngaphandle kwe-ribose eboshwe kwi-E. cuniculi ribosome.Kwi-E. cuniculi ribosome, le nucleotide ithatha indawo efanayo ne-25S rRNA A3186 nucleotide (inombolo ye-Saccharomyces cerevisiae) kwii-ribosomes ezininzi ze-eukaryotic.b Kwisakhiwo se-ribosomal se-E. cuniculi, le nucleotide iphakathi kweeprotheni ze-ribosomal uL9 kunye ne-EL20, ngaloo ndlela izinzile uqhagamshelwano phakathi kweeprotheni ezimbini.cd eL20 ulandelelwano lohlalutyo lolondolozo phakathi kweentlobo ze-microsporidia.Umthi we-phylogenetic weentlobo ze-Microsporidia (c) kunye nolandelelwano oluninzi lwe-eL20 iprotheni (d) ibonisa ukuba i-nucleotide-binding residues F170 kunye ne-K172 zigcinwe kwi-Microsporidia eqhelekileyo, ngaphandle kwe-S. lophii, ngaphandle kwe-branching yokuqala ye-Microsporied egcina i-Microsporidia ye-ES39, egcina i-Microsporidia.e Lo mzobo ubonisa ukuba i-nucleotide-binding residues F170 kunye ne-K172 zikhona kuphela kwi-EL20 ye-microsporidia genome encitshisiweyo kakhulu, kodwa kungekhona kwezinye i-eukaryotes.Ngokubanzi, ezi nkcukacha zibonisa ukuba i-Microsporidian ribosomes yenze indawo yokubopha i-nucleotide ebonakala ibopha i-molecule ye-AMP kwaye iyisebenzise ukuzinzisa i-protein-protein interactions kwi-ribosomal structure.Ukugcinwa okuphezulu kwesi siza esibophelelayo kwi-Microsporidia kunye nokungabikho kwayo kwezinye i-eukaryotes kubonisa ukuba le ndawo inokubonelela ngenzuzo ekhethiweyo yokusinda kwi-Microsporidia.Ke, ipokotho ebophelela i-nucleotide kwi-microsporidia ribosome ayibonakali iyinto ewohlokayo okanye isiphelo sokuthotywa kwe-rRNA njengoko kuchaziwe ngaphambili, kodwa kunoko kuluncedo lwe-evolutionary innovation evumela i-microsporidia ribosome ukuba ibophe ngokuthe ngqo iimolekyuli ezincinci, zisebenzise njengeebhloko zokwakha zemolekyuli.iibhloko zokwakha ribosomes.Oku kufunyenweyo kwenza i-microsporidia ribosome ibe yeyona ribosome eyaziwayo ukusebenzisa i-nucleotide enye njengebhloko yayo yokwakha.f Indlela yokuzivelela ecingelwayo ethathwe kwi-nucleotide ebophelelayo.
Ubunzima besibini be-molecular weight density bufumaneka kwi-interface phakathi kweeprotheyini ze-ribosomal uL9 kunye ne-EL30 (umzobo 5a).Olu jongano lwaluchazwe ngaphambili kwisakhiwo se-Saccharomyces cerevisiae ribosome njengendawo ebophelelayo ye-25S nucleotide ye-rRNA A3186 (inxalenye ye-ES39L rRNA extension)38.Kwaboniswa ukuba kwi-ribosomes ye-P. locustae ES39L ewohlokileyo, olu jongano lubophelela i-nucleotide eyodwa engaziwayo 31, kwaye kucingelwa ukuba le nucleotide yindlela yokugqibela encitshisiweyo ye-rRNA, apho ubude be-rRNA bu- ~ 130-230 iziseko.I-ES39L iyancipha ibe yi-nucleotide enye 32.43.Imifanekiso yethu ye-cryo-EM ixhasa ingcamango yokuba ukuxinana kunokuchazwa ngama-nucleotides.Nangona kunjalo, isisombululo esiphezulu sesakhiwo sethu sibonise ukuba le nucleotide i-molecule ye-extraribosomal, mhlawumbi i-AMP (Umfanekiso 5a, b).
Siye sabuza ukuba ngaba indawo yokubopha i-nucleotide ibonakala kwi-E. cuniculi ribosome okanye ingaba yayikhona ngaphambili.Ekubeni i-nucleotide ebophelelayo ixutywa kakhulu yi-Phe170 kunye ne-Lys172 intsalela kwi-protein ye-ribosomal eL30, siye savavanya ukugcinwa kwezi ntsalela kwi-4396 emele i-eukaryotes.Njengoko kwimeko ye-UL15 ngasentla, sifumene ukuba i-Phe170 kunye ne-Lys172 i-residues zigcinwe kakhulu kuphela kwi-Microsporidia eqhelekileyo, kodwa zingekho kwezinye i-eukaryotes, kuquka i-atypical Microsporidia Mitosporidium kunye ne-Amphiamblys, apho i-ES39L i-rRNA iqhekeza, i-5c 4, i-5c 4, i-5c4, ayincitshisiwe.-e).
Idityaniswe kunye, ezi datha zixhasa ingcamango yokuba i-E. cuniculi kwaye mhlawumbi nezinye i-canonical microsporidia ziye zavelisa amandla okubamba ngokufanelekileyo amanani amakhulu ee-metabolites ezincinci kwisakhiwo se-ribosome ukuhlawulela ukuhla kwe-rRNA kunye namanqanaba eprotheni.Ngokwenza njalo, baye baphuhlisa isakhono esikhethekileyo sokubopha i-nucleotides ngaphandle kwe-ribosome, ebonisa ukuba izakhiwo ze-molekyuli ze-parasitic zihlawulela ngokubamba i-metabolites encinci kwaye zisebenzise njengemilinganiselo yesakhiwo se-RNA ethotyiweyo kunye neeprotheyini zamaqhekeza..
Inxalenye yesithathu engalinganiswanga yemephu yethu ye-cryo-EM, efunyenwe kwi-ribosomal subunit enkulu.Isisombululo esiphezulu (2.6 Å) semephu yethu sicebisa ukuba le ngxinano yeyamaprotheni anendibaniselwano eyodwa yeentsalela ezinkulu zekhonkco elisecaleni, eliye lasivumela ukuba sichonge obu bunzima njengeprotheni ye-ribosomal eyayingaziwa ngaphambili esiyichonge njengeYathiywa igama elithi msL2 (Microsporidia-iprotein ethile ye-L2) (iindlela, umfanekiso 6).Uphando lwethu lwe-homology lubonise ukuba i-msL2 igcinwe kwi-Microsporidia clade ye-genus Encephaliter kunye ne-Orosporidium, kodwa ayikho kwezinye iintlobo, kuquka nezinye i-Microsporidia.Kwisakhiwo se-ribosomal, i-msL2 ithatha i-gap eyenziwe ngokulahleka kwe-ES31L rRNA eyandisiweyo.Kule ndawo, i-msL2 inceda ukuzinzisa ukugoqa kwe-rRNA kwaye inokubuyisela ukulahlekelwa kwe-ES31L (Umfanekiso 6).
Ubuninzi be-Electron kunye nemodeli ye-Microsporidia-specific ribosomal protein msL2 efumaneka kwi-E. cuniculi ribosomes.b Uninzi lwe-eukaryotic ribosomes, kuquka i-80S ribosome ye-Saccharomyces cerevisiae, ine-ES19L rRNA yokukhulisa ilahlekile kwiintlobo ezininzi ze-Microsporidian.Ulwakhiwo olusekwe ngaphambili lwe-V. necatrix microsporidia ribosome lucebisa ukuba ukulahleka kwe-ES19L kwezi zinambuzane kuhlawulwe ngokuvela kweprotein entsha ye-msL1 ribosomal.Kolu phononongo, sifumene ukuba i-E. cuniculi ribosome nayo yavelisa iprotein eyongezelelweyo ye-ribosomal RNA mimic njengembuyekezo ebonakalayo yokulahleka kwe-ES19L.Nangona kunjalo, i-msL2 (okwangoku ichazwe njenge-hypothetical ECU06_1135 protein) kunye ne-msL1 zinemvelaphi eyahlukileyo yolwakhiwo kunye ne-evolution.c Oku kufunyaniswe kokuveliswa kwe-msL1 enganxulumananga kunye ne-msL2 ye-ribosomal proteins icebisa ukuba ukuba ii-ribosomes ziqokelela uguquko oluyingozi kwi-rRNA yazo, zinokufikelela kumanqanaba angazange abonwe ngaphambili eyantlukwano yokuhlanganisa nakwiseti encinci yeentlobo ezinxulumene ngokusondeleyo.Oku kufunyaniswe kunokunceda ukucacisa imvelaphi kunye nokuvela kwe-ribosome ye-mitochondrial, eyaziwa ngokuncipha kwayo kakhulu kwe-rRNA kunye nokwahluka okungaqhelekanga kukwakheka kweeprotheyini kuzo zonke iindidi.
Emva koko sathelekisa iprotheni ye-msL2 kunye neprotheni ye-msL1 echazwe ngaphambili, i-microsporidia-specific ribosomal protein eyaziwayo kuphela efumaneka kwi-V. necatrix ribosome.Besifuna ukuvavanya ukuba i-msL1 kunye ne-msL2 ziyazalana na ngokwendalo.Uhlalutyo lwethu lubonise ukuba i-msL1 kunye ne-msL2 zithatha i-cavity efanayo kwisakhiwo se-ribosomal, kodwa zinezakhiwo ezahlukeneyo eziphambili kunye neendawo eziphezulu, ezibonisa imvelaphi yazo yokuzimela ezimeleyo (umzobo 6).Ke, ukufunyanwa kwethu kwe-msL2 kubonelela ngobungqina bokuba amaqela eentlobo ze-eukaryotic ezixineneyo zinokuzivelela ngokuzimeleyo iiproteni ze-ribosomal ezahlukeneyo ukuhlawula ilahleko yamaqhekeza e-rRNA.Oku kufunyaniswayo kuyaphawuleka ukuba ininzi ye-cytoplasmic eukaryotic ribosomes iqulethe iprotheni engaguqukiyo, kubandakanywa nentsapho efanayo ye-81 ribosomal proteins.Inkangeleko ye-msL1 kunye ne-msL2 kwizigaba ezahlukeneyo ze-microsporidia ekuphenduleni ilahleko yamacandelo e-rRNA eyandisiweyo icebisa ukuba ukuthotywa kwe-architecture ye-molecular ye-parasite kubangela ukuba ii-parasites zifune ukuguqulwa kwembuyekezo, ekugqibeleni okunokuthi kukhokelele ekufumaneni kwazo kwiindawo ezahlukeneyo ze-parasite.izakhiwo.
Ekugqibeleni, xa imodeli yethu igqityiwe, sathelekisa ukubunjwa kwe-E. cuniculi ribosome kunye naleyo yayixelwe kwangaphambili ukusuka kulandelelwano lwegenome.Iiproteni ezininzi ze-ribosomal, kubandakanywa i-eL14, eL38, eL41, kunye ne-eS30, ngaphambili kwakucingelwa ukuba zilahlekile kwi-E. cuniculi genome ngenxa yokungabikho okubonakalayo kwee-homologues zazo kwi-E. cuniculi genome.Ilahleko yeeprotheyini ezininzi ze-ribosomal kukwaqikelelwa ukuba kwezinye iiparasites ze-intracellular ezincitshisiweyo kakhulu kunye ne-endosymbionts.Ngokomzekelo, nangona iibhaktheriya ezininzi ezikhululekile ziqulethe usapho olufanayo lwe-54 yeeprotheyini ze-ribosomal, kuphela i-11 yezi ntsapho zeeprotheyini ezine-homologues ezibonwayo kwi-genome nganye ehlalutyiweyo yebhaktheriya ethintelwe ngumkhosi.Ukuxhasa le ngcamango, ukulahlekelwa kweeprotheni ze-ribosomal kuye kwabonwa ngovavanyo kwi-V. necatrix kunye ne-P. locustae microsporidia, ezingenayo i-eL38 kunye ne-EL4131,32 iiprotheni.
Nangona kunjalo, izakhiwo zethu zibonisa ukuba yi-eL38, eL41, kunye ne-eS30 kuphela ezilahlekileyo kwi-E. cuniculi ribosome.Iprotheyini ye-eL14 igcinwe kwaye isakhiwo sethu sibonise ukuba kutheni le proteni ayifumanekanga kwi-homology search (Umfanekiso 7).Kwi-E. cuniculi ribosomes, uninzi lwendawo yokubopha i-eL14 ilahlekile ngenxa yokuthotywa kwe-rRNA-amplified ES39L.Ukungabikho kwe-ES39L, i-eL14 yalahlekelwa uninzi lwesakhiwo sayo sesibini, kwaye kuphela i-18% yokulandelelana kwe-EL14 yayifana ne-E. cuniculi kunye ne-S. cerevisiae.Oku kugcinwa kokulandelelana okungahambi kakuhle kuyamangalisa kuba i-Saccharomyces cerevisiae kunye ne-Homo sapiens-izinto eziphilayo eziye zavela kwi-1.5 yeminyaka eyibhiliyoni ngokuhlukana-zabelana ngaphezu kwe-51% yeentsalela ezifanayo kwi-EL14.Le lahleko ingaqhelekanga yolondolozo ichaza ukuba kutheni i-E. cuniculi eL14 okwangoku ichazwa njengeproteni yokubeka i-M970_061160 kwaye ingeyiyo i-eL1427 ribosomal protein.
kunye ne-Microsporidia ribosome iphulukene nolwandiso lwe-ES39L rRNA, ethe yaphelisa ngokuyinxenye i-eL14 ribosomal protein binding site.Ukungabikho kwe-ES39L, i-eL14 iprotheni ye-microspore ifumana ukulahlekelwa kwesakhiwo sesibini, apho i-rRNA-binding α-helix yangaphambili iyancipha ibe yincinci yobude obuncinci.b Ulungelelwaniso lolandelelwano oluninzi lubonisa ukuba iprotheyini ye-eL14 igcinwe kakhulu kwiintlobo ze-eukaryotic (i-57% yokulandelelanisa i-identity phakathi kweyeast kunye ne-homologues yabantu), kodwa igcinwe kakubi kwaye iyahlukana kwi-microsporidia (apho kungekho ngaphezu kwe-24% yentsalela efana ne-eL14 homologue).ukusuka S. cerevisiae okanye H. sapiens).Ukugcinwa kokulandelelana okungahambi kakuhle kunye nokwahluka kwesakhiwo sesibini kuchaza ukuba kutheni i-eL14 homologue ayizange ifunyanwe kwi-E. cuniculi kwaye kutheni le proteni kucingelwa ukuba ilahlekile kwi-E. cuniculi.Ngokwahlukileyo koko, i-E. cuniculi eL14 yayikhe yachazwa njengeprotheyini yokubeka i-M970_061160.Olu qwalaselo lubonisa ukuba ukuhlukahluka kwe-microsporidia genome okwangoku kuqikelelwe ngokugqithisileyo: ezinye iijini ezicatshangelwa ukuba zilahlekile kwi-microsporidia enyanisweni zigcinwe, nangona zihluke kakhulu;endaweni yoko, ezinye kucingelwa ukuba ikhowudi microsporidia yofuzo for iiproteni worm-specific (umzekelo, iprotein ecingelwayo M970_061160) eneneni iikhowudi iiproteni ezahlukeneyo kakhulu ezifumaneka kwezinye eukaryotes.
Oku kufunyanisiweyo kuphakamisa ukuba i-rRNA denaturation inokukhokelela kwilahleko enkulu yokugcinwa kolandelelwano kwiiproteni ezikufutshane ne-ribosomal, inika ezi proteni ukuba zingabonakali kuphando lwe-homology.Ngaloo ndlela, sisenokuwuqikelela ngokugqithiseleyo umlinganiselo wokwenyani wokuwohloka kweemolekyuli kwizinto ezincinane zegenome, ekubeni ezinye iiproteni ekucingelwa ukuba zilahlekile zisekho, nangona ziguquguquke kakhulu.
Ii-parasites zinokuwugcina njani umsebenzi woomatshini bazo beemolekyuli phantsi kweemeko zokunciphisa kakhulu i-genome?Uphononongo lwethu luphendula lo mbuzo ngokuchaza ubume bemolekyuli entsonkothileyo (i-ribosome) ye-E. cuniculi, i-organism enenye yezona zincinci ze-eukaryotic genomes.
Kuye kwaziwa malunga neminyaka engamashumi amabini ukuba iiprotheyini kunye neemolekyuli ze-RNA kwii-microbial parasites zihlala zihluke kwii-molekyuli zazo ze-homologous kwiintlobo eziphilayo ezikhululekile ngenxa yokuba zingenamaziko okulawula umgangatho, ziyancipha zibe yi-50% yobukhulu bazo kwii-microbes eziphilayo, njl.iinguqulelo ezininzi eziphazamisayo eziphazamisa ukusongwa kunye nokusebenza.Ngokomzekelo, i-ribosomes yezilwanyana ezincinci ze-genome, ezibandakanya ezininzi ze-intracellular parasites kunye ne-endosymbionts, kulindeleke ukuba zingabi namaprotheni amaninzi e-ribosomal kunye nesinye kwisithathu se-nucleotides ye-rRNA xa kuthelekiswa neentlobo eziphilayo ezikhululekile 27, 29, 30, 49. Nangona kunjalo, indlela ezi molekyuli zisebenza ngayo kwi-mycosymbols efundwayo ngokubanzi, i-mysynomic efundwayo ngokubanzi.
Uphononongo lwethu lubonisa ukuba ubume be-macromolecules bunokutyhila iinkalo ezininzi zokuziphendukela kwemvelo ekunzima ukuzikhupha kwizifundo ze-genomic zokuthelekisa zendabuko ze-intracellular parasites kunye nezinye izinto eziphilayo ezithintelweyo (i-Supplementary Fig. 7).Umzekelo, umzekelo weprotheyini ye-EL14 ubonisa ukuba sinokuyigqithisa iqondo lokwenyani lokuthotywa kwezixhobo zemolekyuli kwiintlobo zeparasitic.I-Encephalic parasites ngoku kukholeleka ukuba inamakhulu ee-microsporidia-specific genes.Nangona kunjalo, iziphumo zethu zibonisa ukuba ezinye zezi ntlobo zemfuza ezibonakala zikhethekileyo ngokwenene zihluke kakhulu kwimizila yemfuza eqhelekileyo kwezinye ii-eukaryotes.Ngaphezu koko, umzekelo weprotheni ye-msL2 ibonisa indlela esingazihoyi ngayo iiproteni ezintsha ze-ribosomal kwaye siwujongela phantsi umxholo woomatshini beemolekyuli.Umzekelo weemolekyuli ezincinci ubonisa indlela esinokungayihoyi ngayo eyona mveliso ikrelekrele kwizakhiwo zemolekyuli ezifunxileyo ezinokuzinika umsebenzi omtsha webhayoloji.
Xa zidibene, ezi ziphumo ziphucula ukuqonda kwethu umahluko phakathi kwezakhiwo zemolekyuli zezinto eziphilayo ezinomda kunye noogxa bazo kwizinto eziphilayo ezikhululekileyo.Sibonisa ukuba oomatshini beemolekyuli, ekukudala kucingwa ukuba bancitshisiwe, bawohloka, yaye baxhomekeke kwiinguqu ezahlukahlukeneyo ezidodobalisayo, endaweni yoko baneseti yezinto ezingaqhelekanga ezingahoywanga ngokucwangcisiweyo.
Kwelinye icala, amaqhekeza e-rRNA angengobuninzi kunye namaqhekeza adityanisiweyo esiwafumene kwi-ribosomes ye-E. cuniculi icebisa ukuba ukuncitshiswa kwegenome kunokutshintsha naloo nxalenye yomatshini osisiseko wemolekyuli ogcinwe kwimida emithathu yobomi - emva kweminyaka ephantse ibe yi-3.5 yezigidigidi.ukuziphendukela kwemvelo okuzimeleyo kweentlobo.
Amaqhekeza e-rRNA angenaqhuma kwaye adityanisiweyo kwi-E. cuniculi ribosomes anomdla ngakumbi ekukhanyeni kwezifundo zangaphambili zeemolekyuli ze-RNA kwiibhaktheriya ze-endosymbiotic.Ngokomzekelo, kwi-aphid endosymbiont ye-Buchnera aphidicola, i-rRNA kunye ne-tRNA iamolekyu zibonakaliswe ukuba zinezakhiwo ezithintekayo kwiqondo lokushisa ngenxa ye-A + T yokwakheka kunye nomlinganiselo ophezulu we-non-canonical base pairs20,50.Olu tshintsho kwi-RNA, kunye neenguqu kwiimolekyuli zeprotheyini, ngoku zicatshangelwa ukuba zijongene nokuxhomekeka kwe-endosymbionts kumaqabane kunye nokungakwazi kwe-endosymbionts ukudlulisa ukushisa kwe-21, i-23.Nangona i-parasitic microsporidia rRNA inotshintsho olwahlukileyo ngokwesakhiwo, ubume bolu tshintsho lubonisa ukuba ukunciphisa ukuzinza kwe-thermal kunye nokuxhomekeka okuphezulu kwiiprotheni ze-chaperone kunokuba ziimpawu eziqhelekileyo ze-molecule ze-RNA kwizinto eziphilayo ezine-genomes ezincitshisiweyo.
Ngakolunye uhlangothi, izakhiwo zethu zibonisa ukuba i-microsporidia ye-parasite iye yavelisa isakhono esikhethekileyo sokuchasa i-rRNA egcinwe ngokubanzi kunye namaqhekeza eprotheni, ukuphuhlisa amandla okusebenzisa i-metabolites encinci kwaye ifumaneka ngokulula njengemilinganiso yesakhiwo se-rRNA kunye namaqhekeza eprotheni.Ukuthotywa kwesakhiwo seemolekyuli..Olu luvo luxhaswa yinyaniso yokuba iimolekyuli ezincinci ezihlawulela ukulahleka kweeprotheyini zamaqhekeza kwi-rRNA kunye ne-ribosomes ye-E. cuniculi ibophelela kwii-microsporidia-specific residues kwi-uL15 kunye ne-eL30 iiprotheni.Oku kuphakamisa ukuba ukubopha iamolekyu ezincinci kwi-ribosomes kunokuba yimveliso yokukhetha okulungileyo, apho ukuguqulwa kwe-Microsporidia-specific in ribosomal proteins kuye kwakhethwa ngenxa yokukwazi ukunyusa ukudibanisa kwe-ribosomes kwiimolekyuli ezincinci, ezinokukhokelela kwizinto eziphilayo ezisebenzayo ze-ribosomal.Ukufunyaniswa kubonakalisa i-smart innovation kwi-molecular structure ye-microbial parasites kwaye isinika ukuqonda okungcono malunga nendlela izakhiwo ze-molecular parasite zigcina umsebenzi wazo nangona i-reductive evolution.
Okwangoku, ukuchongwa kwezi molekyuli ezincinci kuhlala kungacaci.Akucaci ukuba kutheni ukubonakala kwezi molekyuli ezincinci kwisakhiwo se-ribosomal kuhluke phakathi kweentlobo ze-microsporidia.Ngokukodwa, akucaci ukuba kutheni ukubopha i-nucleotide kubonwa kwi-ribosomes ye-E. cuniculi kunye ne-P. locustae, kwaye kungekhona kwi-ribosomes ye-V. necatrix, nangona ubukho be-F170 eseleyo kwi-eL20 kunye ne-K172 iiprotheni ze-V. necatrix.Oku kucinywa kunokubangelwa yintsalela ye-43 uL6 (ebekwe kufuphi nepokotho yokubopha i-nucleotide), eyi-tyrosine kwi-V. necatrix kwaye kungekhona i-threonine kwi-E. cuniculi kunye ne-P. locustae.Ikhonkco elikhulu elinuka kamnandi elisecaleni leTyr43 linokuphazamisana nenucleotide ngenxa yokudityaniswa kwesteric.Ngaphandle koko, ukucinywa kwe-nucleotide okubonakalayo kunokuba ngenxa yesisombululo esisezantsi se-cryo-EM imaging, ethintela ukubunjwa kwe-V. necatrix ribosomal fragments.
Kwelinye icala, umsebenzi wethu ucebisa ukuba inkqubo yokubola kwemfuza isenokuba ngamandla okuvelisa.Ngokukodwa, isakhiwo se-E. cuniculi ribosome sicebisa ukuba ilahleko ye-rRNA kunye namaqhekeza eprotheni kwi-microsporidia ribosome idala uxinzelelo lwendaleko olukhuthaza utshintsho kwisakhiwo se-ribosome.Ezi zantlukwano zenzeka kude nendawo esebenzayo yeribosome kwaye zibonakala zinceda ukugcina (okanye ukubuyisela) eyona indibano yeribosome enokuphazamiseka ngenye indlela yi-rRNA ecuthiweyo.Oku kuphakamisa ukuba into entsha entsha ye-microsporidia ribosome ibonakala ngathi iye yavela kwisidingo sokuthintela ukukhukuliseka kwemfuza.
Mhlawumbi oku kuboniswa kakuhle ngokubophelela kwe-nucleotide, engazange ibonwe kwezinye izinto eziphilayo ukuza kuthi ga ngoku.Inyaniso yokuba i-nucleotide-binding residues ikhona kwi-microsporidia eqhelekileyo, kodwa kungekhona kwezinye i-eukaryotes, ibonisa ukuba iziza ezibophezelayo ze-nucleotide azikho nje i-relics elinde ukunyamalala, okanye indawo yokugqibela ye-rRNA ibuyiselwe kwifom ye-nucleotides nganye.Endaweni yoko, le ndawo ibonakala ngathi yinto eluncedo enokuthi ivele kwimijikelo emininzi yokukhetha okulungileyo.Iziza zokubopha i-Nucleotide zingaba yimveliso yokukhethwa kwendalo: xa i-ES39L ithotyiwe, i-microsporidia inyanzelekile ukuba ifune imbuyekezo yokubuyisela i-ribosome biogenesis efanelekileyo ngokungabikho kwe-ES39L.Ekubeni le nucleotide inokulinganisa unxibelelwano lwe-molecular ye-nucleotide ye-A3186 kwi-ES39L, i-molecule ye-nucleotide iba yibhloko yokwakha ye-ribosome, ukudibanisa kwayo kuphuculwe ngakumbi ngokuguqulwa kokulandelelana kwe-EL30.
Ngokubhekiselele kwi-molecular evolution ye-intracellular parasites, uphononongo lwethu lubonisa ukuba amandla okhetho lwendalo kaDarwin kunye nemfuzo yokubola kwe-genome ayisebenzi ngokufanayo, kodwa i-oscillate.Okokuqala, ukukhukuliseka kwemfuza kuphelisa iimpawu ezibalulekileyo ze-biomolecules, okwenza imbuyekezo ibe yimfuneko kakhulu.Kuphela kuxa izifunxi-gazi ziyanelisa le mfuno ngokhetho lwendalo lukaDarwin apho iimacromolecules zazo ziya kuba nethuba lokuphuhlisa ezona mpawu zinomtsalane neziyilayo.Okubalulekileyo, ukuvela kweziza ze-nucleotide ezibophelelayo kwi-E. cuniculi ribosome icebisa ukuba le pateni yelahleko-yokufumana i-molecular evolution ayipheleli nje ekutshintsheni iinguqu, kodwa ngamanye amaxesha inika imisebenzi emitsha ngokupheleleyo kwi-parasitic macromolecules.
Le ngcamango iyangqinelana nethiyori kaSewell Wright yokulinganisa ukuhamba, ethi inkqubo engqongqo yokhetho lwendalo iyanciphisa isakhono sezinto eziphilayo ekuveliseni51,52,53.Nangona kunjalo, ukuba ukukhukuliseka kwemfuza kuphazamisa ukhetho lwendalo, oku kukhukuliseka kunokuvelisa utshintsho olungaguquguqukiyo ngokwalo (okanye lude lube yingozi) kodwa lukhokelela kwiinguqu ezingaphezulu ezinikezela ukomelela okuphezulu okanye umsebenzi omtsha webhayoloji.Isakhelo sethu sixhasa le ngcamango ngokubonisa ukuba uhlobo olufanayo lokuguqulwa kokunciphisa ukugoba kunye nomsebenzi we-biomolecule ibonakala iyona nto ibangela ukuphuculwa kwayo.Ngokuhambelana ne-win-win evolutionary model, uphando lwethu lubonisa ukuba ukubola kwe-genome, ngokuqhelekileyo kujongwa njengenkqubo ephazamisayo, kukwangumqhubi omkhulu wezinto ezintsha, ngamanye amaxesha kwaye mhlawumbi ngokuphindaphindiweyo ukuvumela i-macromolecules ukuba ifumane imisebenzi emitsha ye-parasitic.unokuzisebenzisa.