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Ukuguquka kweentsholongwane ezibangela iintsholongwane kubandakanya ukuchasana phakathi kokukhethwa kwendalo, okubangela ukuba iintsholongwane ziphucuke, kunye nokuhamba kwezakhi zofuzo, okubangela ukuba iintsholongwane zilahlekelwe ziijini kwaye ziqokelele utshintsho olubi. Apha, ukuze siqonde indlela olu tshintsho oluvela ngayo kwisikali se-macromolecule enye, sichaza ulwakhiwo lwe-cryo-EM lwe-ribosome ye-Encephatozoon cuniculi, into ephilayo ye-eukaryotic ene-genomes encinci kakhulu kwindalo. Ukuncipha okukhulu kwe-rRNA kwi-E. cuniculi ribosomes kuhamba notshintsho olungazange lubonwe ngaphambili, olufana nokuguquka kwee-rRNA linkers ezidibeneyo kunye ne-rRNA ngaphandle kwamaqhuqhuva. Ukongeza, i-E. cuniculi ribosome yasinda ekulahlekelweni kweziqwenga ze-rRNA kunye neeproteni ngokuphuhlisa amandla okusebenzisa iimolekyuli ezincinci njengeemodeli zesakhiwo zeziqwenga ze-rRNA eziwohlokileyo kunye neeproteni. Lilonke, sibonisa ukuba izakhiwo zeemolekyuli ezicingelwa ukuba zinciphile, ziwohloka, kwaye ziphantsi kotshintsho oludodobalisayo zineendlela ezininzi zokuhlawula ezizigcina zisebenza nangona kukho ukuxinana okukhulu kweemolekyuli.
Ngenxa yokuba uninzi lwamaqela ee-microbial parasites anezixhobo ezikhethekileyo ze-molecular zokusebenzisa izidalwa zazo, sihlala kufuneka siphuhlise iindlela ezahlukeneyo zonyango zamaqela ahlukeneyo ee-parasites1,2. Nangona kunjalo, ubungqina obutsha bubonisa ukuba ezinye iinkalo zokuguquka kwe-parasites ziyadibana kwaye ziqikelelwa kakhulu, nto leyo ebonisa isiseko esinokubakho sokungenelela okubanzi kwezonyango kwi-microbial parasites3,4,5,6,7,8,9.
Umsebenzi wangaphambili uchonge umkhwa oqhelekileyo wokuvela kwezinto eziphilayo ezibizwa ngokuba kukunciphisa i-genome okanye ukubola kwe-genome10,11,12,13. Uphando lwangoku lubonisa ukuba xa iintsholongwane zishiya indlela yazo yokuphila ekhululekileyo kwaye zibe ziintsholongwane ezingaphakathi kwiseli (okanye ii-endosymbionts), ii-genome zazo zifumana utshintsho olucothayo kodwa olumangalisayo kwiminyaka eyi-9,11. Kwinkqubo eyaziwa ngokuba kukubola kwe-genome, iintsholongwane ezingaphakathi kwiseli ziqokelela utshintsho olubi oluguqula ii-genes ezininzi ezazibalulekile ngaphambili zibe zii-pseudogenes, nto leyo ekhokelela ekulahlekelweni kancinci kwe-gene kunye nokuwa kwe-mutational14,15. Oku kuwa kunokutshabalalisa ukuya kuthi ga kwi-95% yee-genes kwizinto eziphilayo ezindala ezingaphakathi kwiseli xa kuthelekiswa neentlobo eziphilayo ezikhululekileyo ezinxulumene nazo. Ke ngoko, ukuvela kweentsholongwane ezingaphakathi kwiseli kukulwa phakathi kwamandla amabini achaseneyo: ukhetho lwendalo lukaDarwin, olukhokelela ekuphuculeni iintsholongwane, kunye nokuwa kwe-genome, okuphosa iintsholongwane ekulibalekeni. Indlela i-parasite ekwazile ngayo ukuphuma kule mfazwe yokutsala nokugcina umsebenzi wesakhiwo sayo se-molecular ayikacaci.
Nangona indlela yokubola kwe-genome ingaqondwa ngokupheleleyo, kubonakala ngathi kwenzeka ikakhulu ngenxa yokuguguleka rhoqo kwe-genetic. Ngenxa yokuba iiparasite zihlala kwiindawo ezincinci, ezingahambisaniyo nezesondo, kwaye zinqongophele ngokwe-genetic, azinakuphelisa ngempumelelo utshintsho olubi oluthi ngamanye amaxesha lwenzeke ngexesha lokuphindaphinda kwe-DNA. Oku kukhokelela ekuqokeleleni okungenakulungiseka kwe-genome ye-parasite eyingozi kunye nokunciphisa i-genome ye-parasite. Ngenxa yoko, iparasite ayilahli nje kuphela ii-gene ezingasafunekiyo ukuze iphile kwindawo engaphakathi kweseli. Kukungakwazi kwe-parasite ukuphelisa ngempumelelo utshintsho olubi oluqhubekayo olubangela ukuba olu tshintsho luqokelele kuyo yonke i-genome, kuquka nezona gene zazo zibalulekileyo.
Uninzi lolwazi lwethu lwangoku lokunciphisa i-genome lusekelwe kuphela ekuthelekisweni kwe-genome sequences, ngaphandle kwengqwalasela ingako kutshintsho kwiimolekyuli zokwenyani ezenza imisebenzi yokugcinwa kwendlu kwaye zisebenza njengeethagethi zamayeza ezinokubakho. Izifundo zokuthelekisa zibonise ukuba umthwalo we-delerious intracellular microbial mutations ubonakala ubeka phambili iiproteni kunye ne-nucleic acids ukuba zigobe kwaye zihlangane, nto leyo eyenza ukuba zixhomekeke ngakumbi kwi-chaperone kwaye zibe buthathaka kakhulu kubushushu19,20,21,22,23. Ukongeza, iiparasite ezahlukeneyo—ukuvela okuzimeleyo ngamanye amaxesha okwahlulwa ngeminyaka eyi-2.5 yeebhiliyoni—ziye zalahlekelwa ngokufanayo ngamaziko olawulo lomgangatho kwi-protein synthesis yazo5,6 kunye neendlela zokulungisa i-DNA24. Nangona kunjalo, kuncinci okwaziwayo malunga nempembelelo yendlela yokuphila yangaphakathi kweseli kuzo zonke ezinye iipropati ze-macromolecules zeseli, kubandakanya ukuziqhelanisa ne-molecular ukuya kumthwalo okhulayo we-delerious mutations.
Kulo msebenzi, ukuze siqonde ngcono ukuvela kweeproteni kunye nee-nucleic acids zee-microorganisms zangaphakathi kweseli, sichonge ulwakhiwo lwee-ribosomes ze-parasite yangaphakathi kweseli i-Encephatozoon cuniculi. I-E. cuniculi sisidalwa esifana nesikhunta esikwiqela le-microsporidia ye-parasite ene-genomes ezincinci ngokungaqhelekanga ze-eukaryotic kwaye ke ngoko zisetyenziswa njengezinto eziphilayo ezingumzekelo wokufunda ukubola kwe-genome25,26,27,28,29,30. Kutshanje, ulwakhiwo lwe-ribosome ye-cryo-EM luchongiwe kwii-genomes ezincitshiswe ngokuphakathi ze-Microsporidia, i-Paranosema locustae, kunye ne-Vairimorpha necatrix31,32 (~3.2 Mb genome). Ezi zakhiwo zibonisa ukuba ukulahleka okuthile kokwandiswa kwe-rRNA kuhlawulelwa kukuphuhliswa koqhagamshelwano olutsha phakathi kweeproteni ze-ribosomal ezikufutshane okanye ukufunyanwa kweeproteni ze-ribosomal ezintsha ze-msL131,32. Iintlobo ze-Encephalitozoon (i-genome ~2.5 yezigidi ze-bp), kunye ne-Ordospora esondeleyo, zibonisa inqanaba lokugqibela lokunciphisa i-genome kwi-eukaryotes - zine-genes ezingaphantsi kwama-2000 ze-protein-coding, kwaye kulindeleke ukuba ii-ribosomes zazo azinazo nje kuphela iziqwenga zokwandiswa kwe-rRNA (iziqwenga ze-rRNA ezahlula ii-ribosomes ze-eukaryotic kwi-ribosomes zebhaktheriya) zikwanazo neeproteni ezine ze-ribosomal ngenxa yokungabikho kwee-homologues kwi-genome ye-E. cuniculi26,27,28. Ke ngoko, sigqibe kwelokuba i-ribosome ye-E. cuniculi inokutyhila amaqhinga angaziwa ngaphambili okuziqhelanisa ne-molecular to decay genome.
Ulwakhiwo lwethu lwe-cryo-EM lumele eyona ribosome incinci ye-eukaryotic cytoplasmic echazwe kwaye inika ukuqonda kwindlela inqanaba lokugqibela lokunciphisa i-genome elichaphazela ngayo isakhiwo, ukuhlanganiswa, kunye nokuguquka koomatshini beemolekyuli abayinxalenye yeseli. Sifumanise ukuba i-E. cuniculi ribosome iyaphula imigaqo emininzi egciniweyo yokugoba kwe-RNA kunye nokuhlanganiswa kwe-ribosome, kwaye safumanisa iproteni entsha ye-ribosomal, eyayingaziwa ngaphambili. Ngokungalindelekanga, sibonisa ukuba ii-microsporidia ribosomes ziye zaphuhlisa amandla okubopha iimolekyuli ezincinci, kwaye sicinga ukuba ukunqunyulwa kwi-rRNA kunye neeproteni kubangela ukuveliswa kwezinto ezintsha ezinokuthi ekugqibeleni zinike iimpawu eziluncedo kwi-ribosome.
Ukuze siphucule ukuqonda kwethu ngokuguquka kweeproteni kunye nee-nucleic acids kwizinto eziphilayo ezingaphakathi kwiseli, sigqibe ekubeni sahlukanise ii-spores ze-E. cuniculi kwiinkcubeko zeeseli zezilwanyana ezincancisayo ezisulelekileyo ukuze sicoce ii-ribosomes zazo size sifumanise ulwakhiwo lwale ribosomes. Kunzima ukufumana inani elikhulu le-microsporidia ezibangelwa zizinambuzane kuba i-microsporidia ayinakukhuliswa kwindawo enesondlo. Endaweni yoko, ziyakhula kwaye zizale kuphela ngaphakathi kwiseli yomninimzi. Ke ngoko, ukuze sifumane i-biomass ye-E. cuniculi yokuhlanjululwa kwe-ribosome, sosulele umgca weseli yezintso zezilwanyana ezincancisayo i-RK13 ngee-spores ze-E. cuniculi kwaye sakhulisa ezi seli zinesifo kangangeeveki ezininzi ukuze sivumele i-E. cuniculi ukuba ikhule kwaye yande. Sisebenzisa i-monolayer yeseli enesifo emalunga nesiqingatha semitha yesikwere, sikwazile ukucoca malunga ne-300 mg yee-spores ze-Microsporidia kwaye sizisebenzise ukwahlula ii-ribosomes. Emva koko saphazamisa ii-spores ezicociweyo ngee-glass beads kwaye sahlula ii-ribosomes ezikrwada sisebenzisa i-stepwise polyethylene glycol fractionation ye-lysates. Oku kusivumele ukuba sifumane malunga ne-300 µg yee-ribosomes ze-E. cuniculi eziluhlaza ukuze zihlalutywe.
Emva koko siqokelele imifanekiso ye-cryo-EM sisebenzisa iisampulu ze-ribosome eziphumayo saza sazilungisa ezi mifanekiso sisebenzisa iimaski ezihambelana ne-ribosomal subunit enkulu, intloko ye-subunit encinci, kunye ne-subunit encinci. Ngexesha lale nkqubo, siqokelele imifanekiso yee-ribosomal particles ezimalunga ne-108,000 kunye nemifanekiso ye-cryo-EM ebalwe ngesisombululo se-2.7 Å (Imifanekiso eyongezelelweyo 1-3). Emva koko sisebenzise imifanekiso ye-cryoEM ukwenza imodeli ye-rRNA, i-ribosomal protein, kunye ne-hibernation factor Mdf1 enxulumene ne-E. cuniculi ribosomes (Umzobo 1a, b).
a Ulwakhiwo lwe-ribosome ye-E. cuniculi kwi-complex ene-hibernation factor Mdf1 (pdb id 7QEP). b Imephu ye-hibernation factor Mdf1 enxulumene ne-E. cuniculi ribosome. c Imephu yesakhiwo sesibini ethelekisa i-rRNA efunyenweyo kwiintlobo zeMicrosporidian kwizakhiwo ze-ribosomal ezaziwayo. Iiphaneli zibonisa indawo yee-armplified rRNA fragments (ES) kunye neendawo ezisebenzayo ze-ribosome, kubandakanya indawo yokucacisa (DC), i-sarcinicin loop (SRL), kunye neziko le-peptidyl transferase (PTC). d Uxinano lwe-electron oluhambelana neziko le-peptidyl transferase le-ribosome ye-E. cuniculi lubonisa ukuba le ndawo ye-catalytic inesakhiwo esifanayo kwi-parasite ye-E. cuniculi kunye neehostele zayo, kuquka i-H. sapiens. e, f Uxinano lwee-electron oluhambelanayo lweziko lokucacisa (e) kunye nesakhiwo seskimu seziko lokucacisa (f) zibonisa ukuba i-E. cuniculi ineentsalela ze-U1491 endaweni ye-A1491 (i-E. coli numbering) kwezinye ii-eukaryotes ezininzi. Olu tshintsho lubonisa ukuba i-E. cuniculi inokuba novelwano kwii-antibiotics ezijolise kule ndawo isebenzayo.
Ngokungafaniyo nezakhiwo ezazimiselwe ngaphambili ze-V. necatrix kunye ne-P. locustae ribosomes (zombini izakhiwo zimele usapho olufanayo lwe-microsporidia i-Nosematidae kwaye zifana kakhulu), ii-ribosomes ze-31,32 E. cuniculi zenza iinkqubo ezininzi ze-rRNA kunye nokuqhekeka kweproteni. Ukuguqulwa okungaphezulu (Imifanekiso eyongezelelweyo 4-6). Kwi-rRNA, utshintsho oluphawulekayo lwaluquka ukulahleka ngokupheleleyo kwe-25S rRNA fragment ES12L ekhulisiweyo kunye nokuwohloka okuncinci kwe-h39, h41, kunye ne-H18 helices (Umzobo 1c, Umfanekiso ongezelelweyo 4). Phakathi kweeproteni ze-ribosomal, utshintsho oluphawulekayo lwaluquka ukulahleka ngokupheleleyo kweproteni ye-eS30 kunye nokufinyezwa kweeproteni ze-eL8, eL13, eL18, eL22, eL29, eL40, uS3, uS9, uS14, uS17, kunye ne-eS7 (Imifanekiso eyongeziweyo 4, 5).
Ngoko ke, ukuncipha okukhulu kwee-genomes zeentlobo ze-Encephalotozoon/Ordospora kubonakala kwisakhiwo sazo se-ribosome: Ii-ribosome ze-E. cuniculi zifumana ukulahleka okukhulu komxholo weproteni kwi-eukaryotic cytoplasmic ribosomes ezixhomekeke kwisimo sokwakheka, kwaye azinazo nezo rRNA kunye neziqwenga zeproteni ezigcinwe ngokubanzi kungekuphela nje kwii-eukaryotes, kodwa nakwiindawo ezintathu zobomi. Ulwakhiwo lwe-ribosome ye-E. cuniculi lubonelela ngemodeli yokuqala yeemolekyuli zezi nguqu kwaye lutyhila iziganeko zokuzivelela ezingakhange zijongwe zii-genomics ezithelekiswayo kunye nezifundo zesakhiwo se-biomolecular sangaphakathi kweseli (Umzobo ongezelelweyo 7). Ngezantsi, sichaza ngasinye sezi ziganeko kunye nemvelaphi yazo yokuzivelela kunye nefuthe lazo elinokubakho kumsebenzi we-ribosome.
Emva koko safumanisa ukuba, ukongeza kwiinqumle ezinkulu ze-rRNA, ii-ribosome ze-E. cuniculi zineenguqu ze-rRNA kwenye yeendawo zazo ezisebenzayo. Nangona iziko le-peptidyl transferase le-ribosome ye-E. cuniculi linesakhiwo esifanayo nezinye ii-ribosome ze-eukaryotic (Umzobo 1d), iziko le-decoding lahlukile ngenxa yokwahluka kolandelelwano kwi-nucleotide 1491 (E. coli numbering, Umfanekiso 1e, f). Olu qwalaselo lubalulekile kuba indawo ye-decoding yee-ribosome ze-eukaryotic idla ngokuqulatha iintsalela ze-G1408 kunye ne-A1491 xa kuthelekiswa neentsalela ze-bacterial-type A1408 kunye ne-G1491. Olu tshintsho lusisiseko sovakalelo olwahlukileyo lwee-ribosomes ze-bacterial kunye ne-eukaryotic kusapho lwe-aminoglycoside lwee-antibiotics ze-ribosomal kunye nezinye iimolekyuli ezincinci ezijolise kwindawo ye-decoding. Kwindawo yokuchaza i-E. cuniculi ribosome, intsalela ye-A1491 yatshintshwa yi-U1491, nto leyo enokubangela ukuba kubekho ujongano olukhethekileyo lokubopha iimolekyuli ezincinci ezijolise kule ndawo isebenzayo. Uhlobo olufanayo lwe-A14901 lukwakho nakwezinye ii-microsporidia ezifana ne-P. locustae kunye ne-V. necatrix, nto leyo ebonisa ukuba ixhaphake kakhulu phakathi kweentlobo ze-microsporidia (Umzobo 1f).
Ngenxa yokuba iisampulu zethu ze-ribosome ze-E. cuniculi zahlulwe kwi-spores ezingasebenziyo ngokwe-metabolically, sivavanye imephu ye-cryo-EM ye-E. cuniculi ukuze sibone ukuba i-ribosome binding echazwe ngaphambili injani phantsi kweemeko zoxinzelelo okanye zendlala. Izinto ezibangela ubusika 31,32,36,37, 38. Sifanise isakhiwo esisekwe ngaphambili se-ribosome ebangela ubusika kunye nemephu ye-cryo-EM ye-E. cuniculi ribosome. Kwi-docking, ii-ribosome ze-S. cerevisiae zisetyenzisiwe kwi-complex ene-hibernation factor Stm138, ii-loucher ribosomes kwi-complex ene-Lso232 factor, kunye nee-ribosomes ze-V. necatrix kwi-complex ene-Mdf1 kunye ne-Mdf231 factors. Kwangaxeshanye, sifumene uxinano lwe-cryo-EM oluhambelana ne-rest factor Mdf1. Ngokufana ne-Mdf1 ebopha kwi-ribosome ye-V. necatrix, i-Mdf1 ikwabopha kwi-ribosome ye-E. cuniculi, apho ivala indawo ye-E ye-ribosome, mhlawumbi inceda ekwenzeni ii-ribosome zifumaneke xa ii-spores ze-parasite zingasebenzi ngokwe-metabolic xa umzimba ungasebenzi (Umfanekiso 2).
I-Mdf1 ivimba indawo ye-E ye-ribosome, ebonakala ngathi inceda ekuvaleleni i-ribosome xa ii-spores ze-parasite zingasebenzi ngokwe-metabolic. Kwisakhiwo se-E. cuniculi ribosome, sifumanise ukuba i-Mdf1 yenza unxibelelwano olungaziwa ngaphambili nesiqu se-L1 ribosome, inxalenye ye-ribosome eququzelela ukukhululwa kwe-tRNA e-deacylated kwi-ribosome ngexesha lokwenziwa kweproteni. Olu nxibelelwano lubonisa ukuba i-Mdf1 iyahlukana ne-ribosome isebenzisa indlela efanayo ne-tRNA e-deacetylated, inika ingcaciso enokwenzeka yokuba i-ribosome iyisusa njani i-Mdf1 ukuze ivuselele ukwenziwa kweproteni.
Nangona kunjalo, isakhiwo sethu sityhile unxibelelwano olungaziwayo phakathi kweMdf1 kunye nomlenze we-ribosome ye-L1 (inxalenye ye-ribosome enceda ukukhulula i-tRNA e-deacylated kwi-ribosome ngexesha lokwenziwa kweproteni). Ngokukodwa, i-Mdf1 isebenzisa unxibelelwano olufanayo ne-elbow segment ye-molecule ye-tRNA e-deacylated (Umzobo 2). Olu hlobo lwemodeli ye-molecular olungaziwa ngaphambili lubonise ukuba i-Mdf1 iyahlukana ne-ribosome isebenzisa indlela efanayo ne-tRNA e-deacetylated, echaza indlela i-ribosome eyisusa ngayo le nto yokuqhwalela ukuze ivuselele ukwenziwa kweproteni.
Xa sakha imodeli ye-rRNA, sifumanise ukuba i-E. cuniculi ribosome ineziqwenga ze-rRNA ezisongiweyo ngendlela engaqhelekanga, esizibize ngokuba yi-fused rRNA (Umzobo 3). Kwi-ribosomes ezigubungela iindawo ezintathu zobomi, i-rRNA igoqeka ibe zizakhiwo apho uninzi lwe-rRNA luseka khona nokuba ludibanisa isiseko kwaye lugoqeka okanye lusebenzisane neeproteni ze-ribosomal38,39,40. Nangona kunjalo, kwi-E. cuniculi ribosomes, ii-rRNAs zibonakala ziphula lo mgaqo wokugoqeka ngokuguqula ezinye zeehelices zazo zibe ziingingqi ze-rRNA ezingavulekanga.
Ulwakhiwo lwe-H18 25S rRNA helix kwi-S. cerevisiae, V. necatrix, kunye ne-E. cuniculi. Ngokwesiqhelo, kwiiribosomes ezigubungela iindawo ezintathu zobomi, le linker ijikeleza kwi-RNA helix equlethe iintsalela ezingama-24 ukuya kuma-34. Kwi-Microsporidia, ngokuchaseneyo, le linker ye-rRNA incitshiswa kancinci kancinci ibe ziikhonkco ezimbini ezine-single-stranded uridine-rich linkers eziqulethe iintsalela ezili-12 kuphela. Uninzi lwale ntsalela luvezwa kwi-solvents. Lo mfanekiso ubonisa ukuba i-microsporidia ye-parasitic ibonakala ngathi iyaphula imigaqo-nkqubo yokugoba kwe-rRNA, apho iziseko ze-rRNA zihlala zidityaniswe nezinye iziseko okanye zibandakanyeka kunxibelelwano lwe-rRNA-protein. Kwi-microsporidia, ezinye iziqwenga ze-rRNA zithatha indawo engathandekiyo, apho i-rRNA helix yangaphambili iba yingqokelela ene-stranded enye ende phantse kumgca othe tye. Ubukho bale mimandla ingaqhelekanga buvumela i-microsporidia rRNA ukuba ibophe iziqwenga ze-rRNA ezikude isebenzisa inani elincinci leziseko ze-RNA.
Umzekelo obalaseleyo wolu tshintsho lwendalo unokubonwa kwi-H18 25S rRNA helix (Umzobo 3). Kwiintlobo ukusuka kwi-E. coli ukuya ebantwini, iziseko zale rRNA helix ziqulathe ii-nucleotides ezingama-24-32, zenza i-helix engahambelaniyo kangako. Kwizakhiwo ze-ribosomal ezazichongiwe ngaphambili ezivela kwi-V. necatrix kunye ne-P. locustae,31,32 iziseko ze-H18 helix azijikwanga, kodwa ukudibana kwesiseko se-nucleotide kuyagcinwa. Nangona kunjalo, kwi-E. cuniculi le fragment ye-rRNA iba zezona zikhonkco zimfutshane i-228UUUGU232 kunye ne-301UUUUUUUUU307. Ngokungafaniyo neziqwenga ze-rRNA eziqhelekileyo, ezi zikhonkco zityebileyo ze-uridine azijikwanga okanye zenze unxibelelwano olukhulu neeproteni ze-ribosomal. Endaweni yoko, zamkela izakhiwo ezivulekileyo nezivulelekileyo ngokupheleleyo apho imicu ye-rRNA yoluliwe phantse ngokuthe tye. Olu hlobo lokwakheka olude luchaza indlela i-E. cuniculi esebenzisa ngayo iziseko ze-RNA ezili-12 kuphela ukuzalisa umsantsa we-33 Å phakathi kwe-H16 kunye ne-H18 rRNA helices, ngelixa ezinye iintlobo zifuna ubuncinane iziseko ze-rRNA eziphindwe kabini ukuzalisa umsantsa.
Ngoko ke, singabonisa ukuba, ngokugoba okungathandekiyo ngamandla, ii-microsporidia ezibangelwa zizinambuzane ziye zaphuhlisa icebo lokucutha kwanazo iinxalenye ze-rRNA ezihlala zigcinwe ngokubanzi kwiintlobo ngeentlobo kwiindawo ezintathu zobomi. Ngokucacileyo, ngokuqokelelana kwezakhi zofuzo eziguqula ii-rRNA helices zibe zii-poly-U linkers ezimfutshane, i-E. cuniculi inokwenza iziqwenga ze-rRNA ezingaqhelekanga eziqulathe ii-nucleotides ezimbalwa kangangoko kunokwenzeka ukuze kuhlanganiswe iziqwenga ze-rRNA ezikude. Oku kunceda ukuchaza indlela i-microsporidia efikelele ngayo ekunciphiseni okukhulu kwisakhiwo sayo esisisiseko se-molecular ngaphandle kokulahlekelwa bubume bayo bokwakha kunye nokusebenza.
Olunye uphawu olungaqhelekanga lwe-E. cuniculi rRNA kukuvela kwe-rRNA ngaphandle kokuqina (Umzobo 4). Amaqhuqhuva ziinucleotides ezingenazo ii-base pairs ezijika ziphume kwi-RNA helix endaweni yokufihla kuyo. Uninzi lwee-rRNA protrusions zisebenza njengezincamathelisi zemolekyuli, zinceda ukubopha iiproteni ze-ribosomal ezikufutshane okanye ezinye iziqwenga ze-rRNA. Ezinye zeeqhuqhuva zisebenza njengeehinge, zivumela i-rRNA helix ukuba iguqe kwaye igobe kakuhle ukuze kuveliswe iproteni evelisayo 41.
a I-rRNA protrusion (i-S. cerevisiae numbering) ayikho kwisakhiwo se-ribosome se-E. cuniculi, kodwa ikhona kwezinye ii-eukaryotes ezininzi ze-E. coli, i-S. cerevisiae, i-H. sapiens, kunye ne-E. cuniculi internal ribosomes. iiparasite azinazo ii-bulges ze-rRNA ezindala nezigcinwe kakuhle. Ezi ngqindilili zizinzisa isakhiwo se-ribosome; ngoko ke, ukungabikho kwazo kwi-microsporidia kubonisa ukuzinza okunciphileyo kokugoba kwe-rRNA kwiiparasite ze-microsporidia. Ukuthelekiswa neziqu ze-P (iziqu ze-L7/L12 kwiibhaktheriya) kubonisa ukuba ukulahleka kwee-bumps ze-rRNA ngamanye amaxesha kuhambelana nokuvela kwee-bumps ezintsha ecaleni kwee-bumps ezilahlekileyo. I-H42 helix kwi-23S/28S rRNA ine-bulge yakudala (U1206 kwi-Saccharomyces cerevisiae) eqikelelwa ukuba ineminyaka eyi-3.5 yeebhiliyoni ubudala ngenxa yokukhuselwa kwayo kwiindawo ezintathu zobomi. Kwi-microsporidia, le bulge iyasuswa. Nangona kunjalo, kwavela iqhubu elitsha ecaleni kweqhubu elilahlekileyo (A1306 kwi-E. cuniculi).
Okumangalisayo kukuba, sifumanise ukuba ii-ribosome ze-E. cuniculi azinazo uninzi lwee-bulges ze-rRNA ezifumaneka kwezinye iintlobo, kuquka ii-bulges ezingaphezu kwama-30 ezigcinwe kwezinye ii-eukaryotes (Umzobo 4a). Oku kulahleka kuphelisa unxibelelwano oluninzi phakathi kwee-subunits ze-ribosomal kunye nee-helices ze-rRNA ezikufutshane, ngamanye amaxesha kudala imingxuma emikhulu ngaphakathi kwe-ribosome, okwenza i-ribosome ye-E. cuniculi ibe nemingxuma ngakumbi xa ithelekiswa nee-ribosome zemveli (Umzobo 4b). Okuphawulekayo kukuba, sifumanise ukuba uninzi lwale migqumo yayilahlekile nakwizakhiwo ze-ribosome ze-V. necatrix kunye ne-P. locustae ezazichongiwe ngaphambili, ezazingajongwanga luhlalutyo lwesakhiwo lwangaphambili31,32.
Ngamanye amaxesha ukulahleka kwee-rRNA bulges kuhamba nophuhliso lwee-bulges ezintsha ecaleni kwe-bulge elahlekileyo. Umzekelo, i-ribosomal P-stem ine-U1208 bulge (kwi-Saccharomyces cerevisiae) eyasinda kwi-E. coli ukuya ebantwini kwaye ke ngoko kuqikelelwa ukuba ineminyaka eyi-3.5 yeebhiliyoni ubudala. Ngexesha lokwenziwa kweproteni, le bulge inceda i-P stem ukuba ihambe phakathi kwe-conformations evulekileyo nevaliweyo ukuze i-ribosome ikwazi ukufumana izinto zoguqulelo kwaye izise kwindawo esebenzayo. Kwi-E. cuniculi ribosomes, le bulge ayikho; nangona kunjalo, ukuqina okutsha (G883) okufumaneka kuphela kwiibini ezintathu zesiseko kunokunceda ekubuyiselweni kokuguquguquka okufanelekileyo kwe-P stem (Umzobo 4c).
Idatha yethu kwi-rRNA engenamaqhuqhuva ibonisa ukuba ukunciphisa i-rRNA akupheleli nje ekulahlekelweni kwezinto ze-rRNA kumphezulu we-ribosome, kodwa kunokubandakanya i-nucleus ye-ribosome, okudala isiphene semolekyuli esithile se-parasite esingakhange sichazwe kwiiseli eziphilayo ezikhululekileyo. Iintlobo eziphilayo ziyabonwa.
Emva kokwenza iiproteni ze-ribosomal kunye ne-rRNA, sifumanise ukuba izinto ze-ribosomal eziqhelekileyo azinakuchaza iindawo ezintathu zomfanekiso we-cryo-EM. Ezimbini kwezi ziqwenga ziimolekyuli ezincinci ngobukhulu (Umzobo 5, Umfanekiso ongezelelweyo 8). Inxalenye yokuqala ibekwe phakathi kweeproteni ze-ribosomal uL15 kunye ne-eL18 kwindawo ehlala ihlalwa yi-C-terminus ye-eL18, efinyezwa kwi-E. cuniculi. Nangona singenakukwazi ukumisela ubuni bale molekyuli, ubungakanani kunye nokuma kwesi siqithi soxinano kuchazwe kakuhle bubukho beemolekyuli ze-spermidine. Ukubopha kwayo kwi-ribosome kuzinziswa ziinguqu ezithile ze-microsporidia kwiiproteni ze-uL15 (Asp51 kunye ne-Arg56), ezibonakala ngathi zonyusa ulwalamano lwe-ribosome kule molekyuli incinci, njengoko zivumela i-uL15 ukuba igoqe imolekyuli encinci kwisakhiwo se-ribosomal. Umfanekiso ongezelelweyo 2). 8, idatha eyongezelelweyo 1, 2).
Umfanekiso we-Cryo-EM obonisa ubukho beenucleotides ngaphandle kwe-ribose ebotshelelwe kwi-ribosome ye-E. cuniculi. Kwi-ribosome ye-E. cuniculi, le nucleotide ikwindawo efanayo ne-25S rRNA A3186 nucleotide (i-Saccharomyces cerevisiae numbering) kwezinye ii-ribosomes ezininzi ze-eukaryotic. b Kwisakhiwo se-ribosomal se-E. cuniculi, le nucleotide ikwindawo ephakathi kweeproteni ze-ribosomal uL9 kunye ne-eL20, ngaloo ndlela izinzisa unxibelelwano phakathi kweeproteni ezimbini. cd eL20 uhlalutyo lokugcinwa kwechungechunge phakathi kweentlobo ze-microsporidia. Umthi we-phylogenetic weentlobo zeMicrosporidia (c) kunye nokulungelelaniswa kwe-multiple sequence yeprotheyini ye-eL20 (d) kubonisa ukuba iintsalela ze-nucleotide-binding residues F170 kunye ne-K172 zigcinwe kwiMicrosporidia eqhelekileyo, ngaphandle kwe-S. lophii, ngaphandle kweMicrosporidia yokuqala ekhula, eyagcina ulwandiso lwe-ES39L rRNA. e Lo mfanekiso ubonisa ukuba iintsalela ze-nucleotide-binding residues F170 kunye ne-K172 zikhona kuphela kwi-eL20 ye-genome ye-microsporidia encitshisiweyo kakhulu, kodwa kungekhona kwezinye ii-eukaryotes. Ngokubanzi, ezi datha zibonisa ukuba ii-ribosomes zeMicrosporidian ziye zaphuhlisa indawo yokubopha i-nucleotide ebonakala ibopha iimolekyuli ze-AMP kwaye izisebenzise ukuzinzisa ukusebenzisana kweprotheyini-protein kwisakhiwo se-ribosomal. Ukugcinwa okuphezulu kwale ndawo yokubopha kwiMicrosporidia kunye nokungabikho kwayo kwezinye ii-eukaryotes kubonisa ukuba le ndawo inokubonelela ngenzuzo yokusinda ekhethiweyo kwiMicrosporidia. Ngoko ke, ipokotho yokubopha i-nucleotide kwi-microsporidia ribosome ayibonakali ngathi luphawu oluwohlokayo okanye uhlobo lokugqibela lokuwohloka kwe-rRNA njengoko kuchaziwe ngaphambili, kodwa kunoko lutshintsho oluluncedo lokuzivelela oluvumela i-microsporidia ribosome ukuba ibophe ngokuthe ngqo iimolekyuli ezincinci, izisebenzise njengeebhloko zokwakha iimolekyuli. Olu fumaniso lwenza i-microsporidia ribosome ibe yeyona ribosome kuphela eyaziwayo ukusebenzisa i-nucleotide enye njengebhloko yokwakha yayo. f Indlela yokuzivelela ecingelwayo ephuma kwi-nucleotide binding.
Ubunzima besibini obuphantsi be-molecular bufumaneka kwindawo ephakathi kweeproteni ze-ribosomal uL9 kunye ne-eL30 (Umzobo 5a). Olu jongano lwaluchazwe ngaphambili kwisakhiwo se-Saccharomyces cerevisiae ribosome njengendawo yokubopha i-25S nucleotide ye-rRNA A3186 (inxalenye yolwandiso lwe-ES39L rRNA)38. Kuboniswe ukuba kwi-ribosomes ze-P. locustae ES39L eziwohlokileyo, olu jongano lubopha i-nucleotide enye engaziwayo 31, kwaye kucingelwa ukuba le nucleotide luhlobo lokugqibela oluncitshisiweyo lwe-rRNA, apho ubude be-rRNA buyi ~130-230 bases. I-ES39L incitshisiwe kwi-nucleotide enye 32.43. Imifanekiso yethu ye-cryo-EM ixhasa ingcamango yokuba uxinano lunokuchazwa yi-nucleotides. Nangona kunjalo, isisombululo esiphezulu sesakhiwo sethu sibonise ukuba le nucleotide yi-extraribosomal molecule, mhlawumbi i-AMP (Umzobo 5a, b).
Emva koko sibuze ukuba ingaba indawo yokubopha i-nucleotide ivele kwi-E. cuniculi ribosome okanye yayikhona ngaphambili. Ekubeni ukubopha i-nucleotide kulawulwa kakhulu yi-Phe170 kunye ne-Lys172 residues kwi-eL30 ribosomal protein, sivavanye ukugcinwa kwezi residues kwi-4396 representative eukaryotes. Njengakwimeko ye-uL15 engentla, sifumanise ukuba ii-Phe170 kunye ne-Lys172 residues zigcinwe kakhulu kwi-Microsporidia eqhelekileyo, kodwa azikho kwezinye ii-eukaryotes, kuquka i-atypical Microsporidia Mitosporidium kunye ne-Amphiamblys, apho i-ES39L rRNA fragment ingancitshiswanga 44, 45, 46 (Umzobo 5c). -e).
Xa zizonke, ezi datha zixhasa ingcamango yokuba i-E. cuniculi kunye nezinye ii-microsporidia ezingezizo ezo zichazwe kwi-canonical ziye zaphuhlisa ubuchule bokubamba ngokufanelekileyo inani elikhulu lee-metabolites ezincinci kwisakhiwo se-ribosome ukuze zihlawulele ukwehla kwamanqanaba e-rRNA kunye neeproteni. Ngokwenza njalo, baye baphuhlisa ubuchule obukhethekileyo bokubopha ii-nucleotides ngaphandle kwe-ribosome, nto leyo ebonisa ukuba izakhiwo zeemolekyuli ezibangelwa zi-parasitic zihlawulela ngokubamba ii-metabolites ezincinci ezininzi kwaye zizisebenzise njengee-structural michies ze-RNA kunye neeproteni eziwohlokileyo.
Inxalenye yesithathu engalinganiswanga yemephu yethu ye-cryo-EM, efumaneka kwi-subunit enkulu ye-ribosomal. Isisombululo esiphezulu (2.6 Å) semephu yethu sibonisa ukuba olu density lukwiiproteni ezineendibaniselwano ezikhethekileyo zeentsalela ezinkulu ze-side chain, nto leyo eyasivumela ukuba sichonge olu density njengeproteni ye-ribosomal eyayingaziwa ngaphambili esiyichonge njenge-msL2 (iproteni ethile ye-Microsporidia L2) (iindlela, umfanekiso 6). Uphendlo lwethu lwe-homology lubonise ukuba i-msL2 igcinwe kwi-Microsporidia clade ye-genus Encephaliter kunye ne-Orosporidium, kodwa ayikho kwezinye iintlobo, kuquka nezinye iiMicrosporidia. Kwisakhiwo se-ribosomal, i-msL2 ithatha indawo evulekileyo ngenxa yokulahleka kwe-ES31L rRNA eyandisiweyo. Kule void, i-msL2 inceda ukuzinzisa ukugoba kwe-rRNA kwaye inokuhlawula ukulahleka kwe-ES31L (Umfanekiso 6).
Ubuninzi be-electron kunye nemodeli yeproteni ye-ribosomal ethile ye-Microsporidia i-msL2 efunyenwe kwi-ribosomes ze-E. cuniculi. b Uninzi lwee-ribosomes ze-eukaryotic, kuquka i-ribosome ye-80S ye-Saccharomyces cerevisiae, zine-ES19L rRNA amplification elahlekileyo kwiintlobo ezininzi ze-Microsporidian. Ulwakhiwo olusekwe ngaphambili lwe-V. necatrix microsporidia ribosome lubonisa ukuba ukulahleka kwe-ES19L kwezi parasites kuhlawulelwa kukuguquka kweproteni entsha ye-msL1 ribosomal. Kolu phononongo, sifumanise ukuba i-E. cuniculi ribosome ikwaphuhlise iproteni eyongezelelweyo ye-ribosomal RNA njengembuyekezo ebonakalayo yokulahleka kwe-ES19L. Nangona kunjalo, i-msL2 (okwangoku ichazwa njengeproteni ye-ECU06_1135 ecingelwayo) kunye ne-msL1 zinemvelaphi eyahlukileyo yesakhiwo kunye neyokuvela. Oku kufunyaniswa kokuveliswa kweeproteni ze-ribosomal ze-msL1 kunye ne-msL2 ezingadibaniyo nendaleko kubonisa ukuba ukuba ii-ribosomes ziqokelela utshintsho oluyingozi kwi-rRNA yazo, zinokufikelela kumanqanaba angaqhelekanga okwahluka kwezakhi nakwiqela elincinci leentlobo ezinxulumeneyo. Oku kufunyaniswa kunokunceda ekucaciseni imvelaphi kunye nokuguquka kwe-ribosome ye-mitochondrial, eyaziwa ngokuncipha kakhulu kwe-rRNA kunye nokwahluka okungaqhelekanga kwizakhi zeproteni kuzo zonke iintlobo.
Emva koko sithelekise iproteni ye-msL2 neproteni ye-msL1 echazwe ngaphambili, eyona proteni ye-ribosomal eyaziwayo ye-microsporidia efumaneka kwi-ribosome ye-V. necatrix. Sifuna ukuvavanya ukuba i-msL1 kunye ne-msL2 zinxulumene ngokwendalo na. Uhlalutyo lwethu lubonise ukuba i-msL1 kunye ne-msL2 zihlala kwindawo enye kwisakhiwo se-ribosomal, kodwa zinezakhiwo ezahlukeneyo eziphambili nezesithathu, nto leyo ebonisa imvelaphi yazo ezimeleyo yokuzivelela (Umzobo 6). Ke ngoko, ukufunyanwa kwethu kwe-msL2 kubonelela ubungqina bokuba amaqela eentlobo ze-eukaryotic ezincinci anokuvelisa ngokuzimeleyo iiproteni ze-ribosomal ezahlukeneyo ukuze ahlawulele ukulahleka kweziqwenga ze-rRNA. Olu fumaniso luphawuleka kuba uninzi lwee-ribosomes ze-cytoplasmic eukaryotic ziqulethe iproteni engaguqukiyo, kuquka usapho olufanayo lweeproteni ze-ribosomal ezingama-81. Ukubonakala kwe-msL1 kunye ne-msL2 kwiindidi ezahlukeneyo ze-microsporidia ukuphendula ekulahlekelweni kwamacandelo e-rRNA ande kubonisa ukuba ukonakala koyilo lweemolekyuli zeparasite kubangela ukuba iiparasite zifune utshintsho oluhlawulelwayo, olunokuthi ekugqibeleni lukhokelele ekufumanekeni kwazo kwiindawo ezahlukeneyo zeparasite.
Ekugqibeleni, xa imodeli yethu igqityiwe, sithelekise ukwakheka kwe-ribosome ye-E. cuniculi noko bekuqikelelwe kwi-genome sequence. Iiproteni ezininzi ze-ribosomal, kuquka i-eL14, i-eL38, i-eL41, kunye ne-eS30, ngaphambili kwakucingelwa ukuba azikho kwi-genome ye-E. cuniculi ngenxa yokungabikho okubonakalayo kwee-homologues zazo kwi-genome ye-E. cuniculi. Ukulahleka kweeproteni ezininzi ze-ribosomal kuqikelelwe nakwezinye iiparasite ezingaphakathi kweseli ezinciphileyo kunye nee-endosymbionts. Umzekelo, nangona uninzi lweebhaktheriya eziphilayo ezikhululekileyo ziqulathe usapho olufanayo lweeproteni ze-ribosomal ezingama-54, ziintsapho ezili-11 kuphela zezi protein ezinee-homologues ezibonakalayo kwi-genome nganye ehlalutyiweyo yeebhaktheriya ezithintelweyo ngumninimzi. Ukuxhasa le ngcamango, ukulahleka kweeproteni ze-ribosomal kuye kwabonwa ngovavanyo kwi-V. necatrix kunye ne-P. locustae microsporidia, ezingenazo iiproteni ze-eL38 kunye ne-eL4131,32.
Nangona kunjalo, izakhiwo zethu zibonisa ukuba kuphela i-eL38, i-eL41, kunye ne-eS30 ezilahlekileyo kwi-ribosome ye-E. cuniculi. Iproteni ye-eL14 igciniwe kwaye isakhiwo sethu sibonise ukuba kutheni le proteni ingafumaneki kuphendlo lwe-homology (Umzobo 7). Kwi-ribosomes ze-E. cuniculi, uninzi lwendawo yokubopha ye-eL14 ilahlekile ngenxa yokuwohloka kwe-ES39L ekhuliswe yi-rRNA. Ngaphandle kwe-ES39L, i-eL14 ilahlekelwe luninzi lwesakhiwo sayo sesibini, kwaye yi-18% kuphela yolandelelwano lwe-eL14 olwalufana kwi-E. cuniculi kunye ne-S. cerevisiae. Olu londolozo olubi lolandelelwano luyamangalisa kuba ne-Saccharomyces cerevisiae kunye ne-Homo sapiens—izidalwa eziphilayo ezahlukane ngeminyaka eyi-1.5 yeebhiliyoni—zabelana ngaphezulu kwe-51% yeentsalela ezifanayo kwi-eL14. Oku kulahleka okungaqhelekanga kolondolozo kuchaza isizathu sokuba i-E. cuniculi eL14 okwangoku ichazwa njengeproteni ye-M970_061160 enokuthiwa yi-proteni ye-ribosomal ye-eL1427.
kwaye iMicrosporidia ribosome ilahlekelwe yi-ES39L rRNA extension, eyasusa ngokuyinxenye indawo yokubopha i-eL14 ribosomal protein. Xa kungekho ES39L, iproteni ye-eL14 microspore ilahlekelwa yisakhiwo sesibini, apho i-α-helix yangaphambili ebopha i-rRNA ibola ibe yi-loop encinci yobude. b Ukulungelelaniswa kwe-multiple sequence kubonisa ukuba iproteni ye-eL14 igcinwe kakhulu kwiintlobo ze-eukaryotic (ulwazi lwe-57% sequence phakathi kwe-yeast kunye ne-homologues zabantu), kodwa ayigcinwanga kakuhle kwaye yahluka kwi-microsporidia (apho kungekho ngaphezulu kwe-24% yeentsalela ezifanayo ne-eL14 homologue). ukusuka kwi-S. cerevisiae okanye kwi-H. sapiens). Olu londolozo lwe-sequence olubi kunye nokwahluka kwesakhiwo sesibini kuchaza isizathu sokuba i-eL14 homologue ingazange ifunyanwe kwi-E. cuniculi kwaye kutheni le proteni kucingelwa ukuba ilahlekile kwi-E. cuniculi. Ngokwahlukileyo koko, i-E. cuniculi eL14 yayikhe yachazwa njengeproteni ye-M970_061160. Olu phononongo lubonisa ukuba ukwahluka kwe-genome ye-microsporidia okwangoku kuqikelelwa kakhulu: ezinye ii-genes ezicingelwa ukuba zilahlekile kwi-microsporidia zigcinwe ngokwenene, nangona zikwiindlela ezahlulwe kakhulu; endaweni yoko, ezinye kucingelwa ukuba zibhala ii-genes ze-microsporidia zeeproteni ezithile zeempethu (umz., iproteni ye-M970_061160) ngokwenene ibhala iiproteni ezahlukeneyo kakhulu ezifumaneka kwezinye ii-eukaryotes.
Olu phando lubonisa ukuba ukuguquka kwe-rRNA kunokukhokelela ekulahlekelweni okukhulu kokugcinwa kolandelelwano kwiiproteni ze-ribosomal ezikufutshane, nto leyo eyenza ezi proteni zingafumaneki xa kukhangelwa i-homology. Ke ngoko, sinokuqikelela kakhulu inqanaba lokwenyani lokuwohloka kweemolekyuli kwizinto ezincinci ze-genome, kuba ezinye iiproteni ezicingelwa ukuba zilahlekile ziyaqhubeka, nangona zikwiindlela ezitshintshileyo kakhulu.
Iiparasites zingawugcina njani umsebenzi wee-molecular machines zazo phantsi kweemeko zokunciphisa kakhulu i-genome? Uphononongo lwethu luphendula lo mbuzo ngokuchaza isakhiwo esintsonkothileyo se-molecular (ribosome) se-E. cuniculi, into ephilayo ene-genomes encinci ye-eukaryotic.
Sele kudlule phantse iminyaka engamashumi amabini kwaziwa ukuba iimolekyuli zeproteni kunye ne-RNA kwiparasite eziziintsholongwane zihlala zahlukile kwiimolekyuli zazo eziziintlobo ngeentlobo eziziintsholongwane kuba azinazo iziko lokulawula umgangatho, zincitshiswa zibe yi-50% yobukhulu bazo kwi-free-living microbes, njl.njl. utshintsho oluninzi oludodobalisa ukugoba nokusebenza. Umzekelo, ii-ribosomes zezinto eziphilayo ezincinci ze-genome, kuquka iiparasite ezininzi zangaphakathi kwiseli kunye nee-endosymbionts, kulindeleke ukuba ziswele iiproteni ezininzi ze-ribosomal kunye nesinye kwisithathu see-nucleotides ze-rRNA xa kuthelekiswa neentlobo eziphilayo eziziintsholongwane 27, 29, 30, 49. Nangona kunjalo, indlela ezi molekyuli ezisebenza ngayo kwiparasite ihlala iyimfihlakalo kakhulu, ifundwa ikakhulu nge-genomics yokuthelekisa.
Uphononongo lwethu lubonisa ukuba ulwakhiwo lwee-macromolecules lunokubonisa iinkalo ezininzi zokuvela kwezinto ekunzima ukuzikhupha kwizifundo zemveli zokuthelekisa ii-genomic zeeparasite zangaphakathi kweseli kunye nezinye izinto eziphilayo ezithintelwe ngumninimzi (Umzobo oNcedisayo 7). Umzekelo, umzekelo weproteni ye-eL14 ubonisa ukuba singayigqithisa inqanaba lokwenyani lokuwohloka kwesixhobo se-molecular kwiintlobo ze-parasite. Iiparasite ze-Encephalitic ngoku zikholelwa ukuba zinamakhulu ee-genes ezithile ze-microsporidia. Nangona kunjalo, iziphumo zethu zibonisa ukuba ezinye zezi genes zibonakala ngathi zichanekile ngokwenene ziintlobo ezahlukeneyo ze-genes eziqhelekileyo kwezinye ii-eukaryotes. Ngaphezu koko, umzekelo weproteni ye-msL2 ubonisa indlela esizityeshela ngayo iiproteni ezintsha ze-ribosomal kwaye sizityeshele umxholo wee-molecular machines ze-parasite. Umzekelo wee-molecules ezincinci ubonisa indlela esinokuzityeshela ngayo izinto ezintsha ezinobuchule kwizakhiwo ze-molecular ze-parasite ezinokuzinika umsebenzi omtsha webhayoloji.
Xa zizonke, ezi ziphumo ziphucula ukuqonda kwethu umahluko phakathi kwezakhiwo zeemolekyuli zezinto eziphilayo ezithintelweyo ngumninimzi kunye nezinye izinto eziphilayo ezikhululekileyo. Sibonisa ukuba oomatshini beemolekyuli, ababekade becinga ukuba banciphile, bayawohloka, kwaye baphantsi kotshintsho olwahlukeneyo oludodobalisayo, endaweni yoko baneempawu zesakhiwo ezingaqhelekanga ezingajongwanga ngendlela ecwangcisiweyo.
Kwelinye icala, iziqwenga ze-rRNA ezingezizo ezinkulu kunye neziqwenga ezidityanisiweyo esizifumene kwi-ribosomes ze-E. cuniculi zibonisa ukuba ukunciphisa i-genome kunokutshintsha kwanazo ezo ndawo zoomatshini beemolekyuli ezisisiseko ezigcinwe kwiindawo ezintathu zobomi - emva kweminyaka ephantse ibe yi-3.5 yeebhiliyoni.
Iziqwenga ze-rRNA ezingenamaqhuqhuva kunye nezihlanganisiweyo kwi-ribosomes ze-E. cuniculi zinomdla kakhulu ekukhanyeni kwezifundo zangaphambili zeemolekyuli ze-RNA kwibhaktheriya ye-endosymbiotic. Umzekelo, kwi-aphid endosymbiont Buchnera aphidicola, iimolekyuli ze-rRNA kunye ne-tRNA zibonakalisiwe zinezakhiwo ezibuthathaka kubushushu ngenxa yokukhetha ukwakheka kwe-A+T kunye nenani eliphezulu leepair zesiseko ezingezizo ze-canonical20,50. Olu tshintsho kwi-RNA, kunye notshintsho kwiimolekyuli zeproteni, ngoku kucingelwa ukuba luxanduva lokuxhomekeka kakhulu kwee-endosymbionts kumaqabane kunye nokungakwazi kwee-endosymbionts ukudlulisa ubushushu 21, 23. Nangona i-parasitic microsporidia rRNA inotshintsho olwahlukileyo ngokwesakhiwo, uhlobo lwale tshintsho lubonisa ukuba ukuzinza kobushushu obuphantsi kunye nokuxhomekeka okuphezulu kwiiproteni ze-chaperone kunokuba ziimpawu eziqhelekileyo zeemolekyuli ze-RNA kwizinto eziphilayo ezinee-genomes ezinciphileyo.
Kwelinye icala, izakhiwo zethu zibonisa ukuba i-parasite microsporidia iphuhlise amandla awodwa okumelana neziqwenga ze-rRNA kunye neeproteni ezigciniweyo ngokubanzi, iphuhlisa amandla okusebenzisa ii-metabolites ezincinci ezininzi nezifumaneka lula njengeemodeli zesakhiwo se-rRNA ewohlokileyo kunye neziqwenga zeproteni. Ukuwohloka kwesakhiwo se-molecular. . Olu luvo luxhaswa yinto yokuba ii-molecules ezincinci ezihlawulela ukulahleka kweziqwenga zeproteni kwi-rRNA kunye nee-ribosomes ze-E. cuniculi zibopha kwiintsalela ezithile ze-microsporidia kwiiproteni ze-uL15 kunye ne-eL30. Oku kuthetha ukuba ukubopha ii-molecules ezincinci kwi-ribosomes kunokuba yimveliso yokukhetha okuhle, apho utshintsho oluthile lwe-Microsporidia kwiiproteni ze-ribosomal lukhethwe ngenxa yokukwazi kwazo ukwandisa ulwalamano lwee-ribosomes kwiimolekyuli ezincinci, okunokukhokelela kwizidalwa ze-ribosomal ezisebenzayo ngakumbi. Oku kufunyanisiweyo kutyhila ubuchule obukrelekrele kwisakhiwo se-molecular se-microbial parasite kwaye kusinika ukuqonda okungcono kwendlela izakhiwo ze-parasite molecular ezigcina ngayo umsebenzi wazo nangona kukho ukuvela okuncitshiswayo.
Okwangoku, ukuchongwa kwezi molekyuli zincinci akukacaci. Akucaci ukuba kutheni ukubonakala kwezi molekyuli zincinci kwisakhiwo se ribosomal kwahlukile kwiintlobo ze microsporidia. Ngokukodwa, akucaci ukuba kutheni ukubopha kwe nucleotide kubonwa kwi ribosomes ze E. cuniculi kunye ne P. locustae, kungekhona kwi ribosomes ze V. necatrix, nangona kukho intsalela ye F170 kwi eL20 kunye ne K172 proteins ze V. necatrix. Oku kususwa kunokubangelwa yi residue 43 uL6 (ebekwe ecaleni kwe nucleotide binding pocket), eyi tyrosine kwi V. necatrix kwaye ingabi yi threonine kwi E. cuniculi kunye ne P. locustae. I chain enkulu ye aromatic side ye Tyr43 inokuphazamisana nokubopha kwe nucleotide ngenxa yokudibana kwe steric. Ngaphandle koko, ukususwa kwe nucleotide okubonakalayo kunokubangelwa sisisombululo esiphantsi se cryo-EM imaging, esithintela ukumodelwa kweziqwenga ze ribosomal ze V. necatrix.
Kwelinye icala, umsebenzi wethu ubonisa ukuba inkqubo yokubola kwe-genome inokuba ngamandla asungula izinto ezintsha. Ngokukodwa, ulwakhiwo lwe-E. cuniculi ribosome lubonisa ukuba ukulahleka kwe-rRNA kunye neziqwenga zeproteni kwi-microsporidia ribosome kudala uxinzelelo lokuzivelela olukhuthaza utshintsho kwisakhiwo se-ribosome. Ezi ntlobo zenzeka kude nendawo esebenzayo ye-ribosome kwaye zibonakala zinceda ukugcina (okanye ukubuyisela) indibano ye-ribosome efanelekileyo ebingaphazanyiswa yi-rRNA enciphisiweyo. Oku kubonisa ukuba uyilo olukhulu lwe-microsporidia ribosome lubonakala ngathi luguqukele kwisidingo sokuthintela ukusasazeka kwezakhi zofuzo.
Mhlawumbi oku kuboniswa ngcono kukubopha kwe-nucleotide, okungazange kubonwe kwezinye izinto eziphilayo ukuza kuthi ga ngoku. Inyani yokuba iintsalela ze-nucleotide-binding zikhona kwi-microsporidia eqhelekileyo, kodwa kungekhona kwezinye ii-eukaryotes, ibonisa ukuba iindawo ze-nucleotide-binding azizo nje ii-relics ezilindele ukunyamalala, okanye indawo yokugqibela yokuba i-rRNA ibuyiselwe kwimo yee-nucleotides nganye. Endaweni yoko, le ndawo ibonakala ngathi luphawu oluluncedo olunokuthi luvele kwiindidi ezininzi zokukhetha okulungileyo. Iindawo ze-Nucleotide binding zinokuba yimveliso yokukhetha kwendalo: xa i-ES39L yonakele, ii-microsporidia zinyanzelwa ukuba zifune imbuyekezo yokubuyisela i-ribosome biogenesis efanelekileyo xa kungekho ES39L. Ekubeni le nucleotide inokulinganisa uqhagamshelwano lweemolekyuli ze-A3186 nucleotide kwi-ES39L, i-nucleotide molecule iba sisakhiwo se-ribosome, ukubopha kwayo kuphuculwe ngakumbi ngokuguqulwa kolandelelwano lwe-eL30.
Ngokuphathelele ukuguquka kweemolekyuli zeeparasite zangaphakathi kweseli, uphando lwethu lubonisa ukuba amandla okukhethwa kwendalo kukaDarwin kunye nokuguquka kwezakhi zofuzo kokubola kwe-genome azisebenzi ngaxeshanye, kodwa ziyatshintshatshintsha. Okokuqala, ukuguquka kwezakhi zofuzo kususa iimpawu ezibalulekileyo zee-biomolecules, okwenza ukuba imbuyekezo ifuneke kakhulu. Kuphela xa iiparasite zanelisa le mfuno ngokukhethwa kwendalo kukaDarwin apho ii-macromolecules zazo zinokufumana ithuba lokuphuhlisa iimpawu zazo ezimangalisayo nezintsha. Okubalulekileyo kukuba, ukuguquka kweendawo zokubopha i-nucleotide kwi-E. cuniculi ribosome kubonisa ukuba le patheni yokuguquka kweemolekyuli elahlekileyo ukuya ekuzuzeni ayipheleli nje ekupheliseni utshintsho olubi, kodwa ngamanye amaxesha inika imisebenzi emitsha ngokupheleleyo kwii-macromolecules ezidla ngokudla.
Le ngcinga ihambelana nethiyori kaSewell Wright yokulingana okuhambayo, ethi inkqubo engqongqo yokukhetha kwendalo ithintela amandla ezinto eziphilayo okuvelisa izinto ezintsha51,52,53. Nangona kunjalo, ukuba ukushukuma kwezakhi zofuzo kuphazamisa ukhetho lwendalo, ezi ntshukumo zinokubangela utshintsho olungaziguquguqukiyo ngokwazo (okanye olunokonakalisa) kodwa olukhokelela kutshintsho olongezelelekileyo olubonelela ngokuqina okuphezulu okanye umsebenzi omtsha webhayoloji. Isakhelo sethu sixhasa le ngcinga ngokubonisa ukuba uhlobo olufanayo loguquko olunciphisa ukugoba kunye nomsebenzi we-biomolecule lubonakala luyinto ephambili yokuphucula kwayo. Ngokuhambelana nemodeli yokuguquka kwe-win-win, uphando lwethu lubonisa ukuba ukubola kwe-genome, ngokwesiko kujongwa njengenkqubo yokuwohloka, kukwangumqhubi omkhulu wokuvelisa izinto ezintsha, ngamanye amaxesha kwaye mhlawumbi rhoqo kuvumela ii-macromolecules ukuba zenze imisebenzi emitsha ye-parasitic. zinokuzisebenzisa.