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Ko te whanaketanga o ngā pirinoa moroiti he whakahē i waenga i te kōwhiringa taiao, e whakapai ake ai ngā pirinoa, me te nekehanga ira, e ngaro ai ngā ira o ngā pirinoa, e kohikohi ai i ngā whakarerekētanga kino. I konei, kia mārama ai ki te āhua o tēnei whakahē i runga i te tauine o tētahi macromolecule kotahi, ka whakaahuahia e mātou te hanganga cryo-EM o te ribosome o Encephalitozoon cuniculi, he rauropi eukaryotic me tētahi o ngā ira iti rawa o te taiao. Ko te whakahekenga nui o te rRNA i roto i ngā ribosome E. cuniculi e haere tahi ana me ngā huringa hanganga kāore i kitea i mua, pērā i te whanaketanga o ngā hononga rRNA kua honoa me te rRNA kāore he pupuhi. Hei tāpiri, i ora te ribosome E. cuniculi i te ngaronga o ngā kongakonga rRNA me ngā pūmua mā te whakawhanake i te kaha ki te whakamahi i ngā ngota iti hei tauira hanganga o ngā kongakonga rRNA me ngā pūmua kua pakaru. I te nuinga, ka whakaatuhia e mātou ko ngā hanganga ngota i whakaarohia kua roa e whakaitihia ana, kua pirau, ā, kei raro i ngā whakarerekētanga ngoikore he maha ngā tikanga utu e pupuri ana i a rātou kia kaha ahakoa ngā whakahekenga ngota tino nui.
Nā te mea he maha ngā rōpū pirinoa moroiti he taputapu ngota ahurei hei whakamahi i ō rātou kaihautū, he maha ngā wā me whakawhanake e tātou he rongoā rerekē mō ngā rōpū pirinoa rerekē1,2. Heoi, e kī ana ngā taunakitanga hou he ōrite ētahi āhuatanga o te whanaketanga pirinoa, ā, he maha ngā wā ka taea te matapae, e tohu ana i tētahi pūtake pea mō ngā wawaotanga rongoā whānui i roto i ngā pirinoa moroiti3,4,5,6,7,8,9.
Kua tautuhia e ngā mahi o mua tētahi ia o te whanaketanga noa i roto i ngā pirinoa moroiti e kiia nei ko te whakaiti i te ira, te pirau rānei o te ira10,11,12,13. E whakaatu ana ngā rangahau o nāianei, ina whakarērea e ngā moroiti tō rātou momo noho noa, ka huri hei pirinoa ā-roto (ngā endosymbionts rānei), ka puhoi engari ka miharo te hurihanga o ō rātou ira i roto i ngā miriona tau9,11. I roto i tētahi tukanga e mōhiotia ana ko te pirau ira, ka kohikohia e ngā pirinoa moroiti ngā whakarerekētanga kino e huri ana i ngā ira nui o mua hei ira-pērā, ka arahi ki te ngaronga āta o ngā ira me te hinganga o te whakarerekētanga14,15. Ka taea e tēnei hinganga te whakangaro i te 95% o ngā ira i roto i ngā rauropi ā-roto tawhito rawa atu ki ngā momo ora noa e tata ana. Nō reira, ko te whanaketanga o ngā pirinoa ā-roto he kukume pakanga i waenganui i ngā kaha e rua e whakahē ana: te kōwhiringa taiao Darwinian, e arahi ana ki te whakapai ake i ngā pirinoa, me te hinganga o te ira, e maka ana i ngā pirinoa ki te warewaretanga. Kāore anō kia mārama me pēhea te kaha o te pirinoa ki te puta mai i tēnei kukume pakanga me te pupuri i te mahi o tōna hanganga ngota.
Ahakoa kāore anō kia tino mārama te tikanga o te pirau o te ira, te āhua nei nā te maha o ngā ira ira i puta ai. Nā te mea e noho ana ngā pirinoa i roto i ngā taupori iti, kore ira, me ngā taupori iti o te ira, kāore e taea e rātou te whakakore i ngā whakarerekētanga kino e puta ana i ētahi wā i te wā o te tāruarua DNA. Ka arahi tēnei ki te kohikohinga kore e taea te whakakore i ngā whakarerekētanga kino me te whakaiti i te ira pirinoa. Nō reira, ehara i te mea ko ngā ira anake kāore e hiahiatia ana mō tōna oranga i roto i te taiao ā-roto te ngaronga o te pirinoa. Ko te kore e taea e ngā taupori pirinoa te whakakore i ngā whakarerekētanga kino ā-wāhanga te take e kohikohi ai ēnei whakarerekētanga puta noa i te ira, tae atu ki ō rātou ira tino nui.
Ko te nuinga o tō tātou māramatanga o nāianei mō te whakaiti i te ira e ahu mai ana i ngā whakatauritenga o ngā raupapatanga ira, me te iti ake o te aro ki ngā huringa o ngā ngota tūturu e mahi ana i ngā mahi tiaki me te mahi hei whāinga rongoā pea. Kua whakaatuhia e ngā rangahau whakataurite ko te taumahatanga o ngā whakarerekētanga moroiti ā-roto kino te āhua nei ka meinga ngā pūmua me ngā waikawa karihi kia hē te takai me te whakakotahi, ka nui ake te whakawhirinaki ki te chaperone me te tino aro ki te wera19,20,21,22,23. Hei tāpiri, he maha ngā pirinoa—he whanaketanga motuhake i ētahi wā ka wehea e te 2.5 piriona tau—i pāngia e te ngaronga rite o ngā pokapū whakahaere kounga i roto i ā rātou hanganga pūmua5,6 me ngā tikanga whakatika DNA24. Heoi, he iti noa te mōhiotanga mō te pānga o te noho ā-roto ki ngā āhuatanga katoa o ngā macromolecules pūtau, tae atu ki te urutau ngota ki te taumahatanga piki haere o ngā whakarerekētanga kino.
I roto i tēnei mahi, kia pai ake ai te mārama ki te whanaketanga o ngā pūmua me ngā waikawa karihi o ngā moroiti ā-roto, i whakatauhia e mātou te hanganga o ngā ribosome o te pirinoa ā-roto Encephalitozoon cuniculi. Ko E. cuniculi he rauropi rite-haero nō tētahi rōpū microsporidia pirinoa he iti rawa ngā ira eukaryotic, nō reira ka whakamahia hei tauira rauropi hei ako i te pirau o te ira25,26,27,28,29,30. Nō nā tata nei, i whakatauhia te hanganga ribosome cryo-EM mō ngā ira iti o Microsporidia, Paranosema locustae, me Vairimorpha necatrix31,32 (~3.2 Mb ira). E kī ana ēnei hanganga ka utua ētahi ngaronga o te whakanui rRNA mā te whanaketanga o ngā hononga hou i waenga i ngā pūmua ribosomal tata, te whiwhinga rānei o ngā pūmua ribosomal msL131,32 hou. Ko ngā momo Encephalitozoon (ira ~2.5 miriona bp), me tō rātou whanaunga tata ko Ordospora, e whakaatu ana i te taumata mutunga o te whakahekenga ira i roto i ngā eukaryote – he iti iho i te 2000 ngā ira e whakakōwae ana i te pūmua, ā, e tika ana kia kore noa iho ā rātou ribosome i ngā kongakonga whānui rRNA (ngā kongakonga rRNA e wehewehe ana i ngā ribosome eukaryotic mai i ngā ribosome kitakita) ā, e whā hoki ā rātou pūmua ribosomal nā te korenga o ngā homologue i roto i te ira E. cuniculi26,27,28. Nō reira, i whakatau mātou ka taea e te ribosome E. cuniculi te whakaatu i ngā rautaki kāore i te mōhiotia i mua mō te urutau ngota ki te pirau ira.
Ko tā mātou hanganga cryo-EM te ribosome cytoplasmic eukaryotic iti rawa hei whakaahua, ā, e whakarato ana i te māramatanga ki te pānga o te taumata whakamutunga o te whakaiti ira ki te hanganga, te huihuinga, me te whanaketanga o te mīhini ngota e whai wāhi nui ana ki te pūtau. I kitea e mātou ko te ribosome E. cuniculi e takahi ana i te maha o ngā mātāpono kua tiakina whānuitia o te takai RNA me te huihuinga ribosome, ā, i kitea he pūmua ribosomal hou, kāore i te mōhiotia i mua. I te mea kāore i te tumanakohia, ka whakaatuhia e mātou kua whanakehia e ngā ribosome microsporidia te kaha ki te here i ngā ngota iti, ā, e whakapae ana ko ngā tapahi i roto i te rRNA me ngā pūmua ka whakaoho i ngā auahatanga whanaketanga ka taea te hoatu i ngā āhuatanga whai hua ki te ribosome.
Hei whakapai ake i tō mātou māramatanga ki te whanaketanga o ngā pūmua me ngā waikawa karihi i roto i ngā rauropi ā-roto, i whakatau mātou ki te wehe i ngā pua E. cuniculi mai i ngā ahurea o ngā pūtau mammal kua pangia kia purea ai ō rātou ribosome me te whakatau i te hanganga o ēnei ribosome. He uaua ki te whiwhi i te maha o ngā microsporidia pirinoa nā te mea kāore e taea te whakatipu i ngā microsporidia i roto i te reo matūkai. Engari, ka tipu, ka whakaputa uri anake i roto i te pūtau manaaki. Nō reira, hei whiwhi i te koiora E. cuniculi mō te purenga ribosome, i pangia e mātou te raina pūtau whatukuhu mammal RK13 ki ngā pua E. cuniculi, ā, i whakatupuria ēnei pūtau kua pangia mō ētahi wiki kia taea ai e E. cuniculi te tipu me te whakanui. Mā te whakamahi i tētahi paparanga pūtau kua pangia he tata ki te haurua mita tapawha, i taea e mātou te purea tata ki te 300 mg o ngā pua Microsporidia me te whakamahi hei wehe i ngā ribosome. Kātahi ka wetewetehia e mātou ngā pua kua purea ki ngā pirepire karāhe, ā, ka wehea ngā ribosome mata mā te whakamahi i te wehenga polyethylene glycol i ia taahiraa o ngā lysates. Nā tēnei i taea ai e mātou te whiwhi tata ki te 300 µg o ngā ribosome E. cuniculi mata mō te tātari hanganga.
I muri mai ka kohia e mātou ngā whakaahua cryo-EM mā te whakamahi i ngā tauira ribosome i puta mai, ā, ka tukatukahia ēnei whakaahua mā te whakamahi i ngā kopare e rite ana ki te waeine ribosomal nui, te pane waeine iti, me te waeine iti. I roto i tēnei tukanga, i kohia e mātou ngā whakaahua o te 108,000 ngā matūriki ribosomal, ā, i tatauhia ngā whakaahua cryo-EM me te taumira o te 2.7 Å (Ngā Pikitia Tāpiri 1-3). I muri mai ka whakamahia e mātou ngā whakaahua cryoEM hei tauira i te rRNA, te pūmua ribosomal, me te hibernation factor Mdf1 e hono ana ki ngā ribosome E. cuniculi (Pikitia 1a, b).
a Te hanganga o te ribosome E. cuniculi e hono ana ki te hibernation factor Mdf1 (pdb id 7QEP). b Te mahere o te hibernation factor Mdf1 e hono ana ki te ribosome E. cuniculi. c Te mahere hanganga tuarua e whakataurite ana i te rRNA kua whakahokia mai i roto i ngā momo Microsporidian ki ngā hanganga ribosomal e mōhiotia ana. E whakaatu ana ngā panera i te wāhi o ngā maramara rRNA (ES) kua whakanuia me ngā pae hohe ribosome, tae atu ki te pae wetewaehere (DC), te koropiko sarcinicin (SRL), me te pokapū peptidyl transferase (PTC). d Ko te kiato irahiko e rite ana ki te pokapū peptidyl transferase o te ribosome E. cuniculi e kī ana he rite te hanganga o tēnei pae whakatere i roto i te pirinoa E. cuniculi me ōna kaihautū, tae atu ki a H. sapiens. e, f Ko te kiato irahiko e rite ana o te pokapū wetewaehere (e) me te hanganga kauwhata o te pokapū wetewaehere (f) e tohu ana kei a E. cuniculi ngā toenga U1491 hei utu mō te A1491 (te tau o E. coli) i roto i te maha atu o ngā eukaryote. E kī ana tēnei panonitanga he tairongo pea a E. cuniculi ki ngā paturopi e aro ana ki tēnei pae hohe.
He rerekē ki ngā hanganga o mua o ngā ribosome V. necatrix me P. locustae (ko ngā hanganga e rua e tohu ana i te whānau microsporidia kotahi a Nosematidae, ā, he tino ōrite tetahi ki tetahi), 31,32 Ka pāngia ngā ribosome E. cuniculi e ngā tukanga maha o te rRNA me te wehewehenga pūmua. Te whakakorenga anō (Ngā Pikitia Tāpiri 4-6). I roto i te rRNA, ko ngā huringa tino kitea ko te ngaronga katoa o te wāhanga 25S rRNA kua whakanuia ES12L me te pirau wāhanga o ngā helices h39, h41, me H18 (Pikitia 1c, Pikitia Tāpiri 4). I roto i ngā pūmua ribosomal, ko ngā huringa tino kitea ko te ngaronga katoa o te pūmua eS30 me te whakapoto o ngā pūmua eL8, eL13, eL18, eL22, eL29, eL40, uS3, uS9, uS14, uS17, me eS7 (Ngā Pikitia Tāpiri 4, 5).
Nō reira, ko te whakahekenga nui o ngā ira o ngā momo Encephalotozoon/Ordospora e whakaatahia ana i roto i tō rātou hanganga ribosome: Ko ngā ribosome E. cuniculi te mea tino ngaro o te ihirangi pūmua i roto i ngā ribosome cytoplasmic eukaryotic e pāngia ana e te āhuatanga hanganga, ā, kāore i a rātou aua maramara rRNA me te pūmua e tiakina whānuitia ana ehara i te mea i roto i ngā eukaryote anake, engari i roto hoki i ngā rohe e toru o te ora. Ko te hanganga o te ribosome E. cuniculi e whakarato ana i te tauira ngota tuatahi mō ēnei huringa, ā, e whakaatu ana i ngā kaupapa whanaketanga kua warewarehia e ngā ira whakataurite me ngā rangahau mō te hanganga biomolecular intracellular (Pikitia Tāpiri 7). Kei raro nei, ka whakaahuahia e mātou ia o ēnei kaupapa me ō rātou takenga whanaketanga pea me ō rātou pānga pea ki te mahi ribosome.
I kitea e mātou, i tua atu i ngā tapahi rRNA nui, he rerekētanga rRNA kei ngā ribosome E. cuniculi i tētahi o ō rātou pae hohe. Ahakoa he rite te hanganga o te pokapū peptidyl transferase o te ribosome E. cuniculi ki ētahi atu ribosome eukaryotic (Pikitia 1d), he rerekē te pokapū wetewaehere nā te rerekētanga raupapatanga i te nucleotide 1491 (E. coli numbering, Pikitia 1e, f). He mea nui tēnei kitenga nā te mea kei roto i te pae wetewaehere o ngā ribosome eukaryotic ngā toenga G1408 me A1491 ki ngā toenga momo huakita A1408 me G1491. Ko tēnei rerekētanga te pūtake o te rerekētanga o te aro o ngā ribosome huakita me te eukaryotic ki te whānau aminoglycoside o ngā paturopi ribosomal me ētahi atu ngota iti e aro ana ki te pae wetewaehere. I te pae wetewaehere o te ribosome E. cuniculi, i whakakapia te toenga A1491 ki te U1491, ka taea te hanga i tētahi atanga here ahurei mō ngā ngota iti e aro ana ki tēnei pae hohe. Kei roto anō hoki i ētahi atu microsporidia pērā i te P. locustae me te V. necatrix te momo A14901, e whakaatu ana he whānuitia tēnei i waenga i ngā momo microsporidia (Pikitia 1f).
Nā te mea i wehea ā mātou tauira ribosome E. cuniculi mai i ngā pua kore mahi o te pūnaha io, i whakamatauria e mātou te mahere cryo-EM o E. cuniculi mō te herenga ribosome kua whakaahuatia i mua i raro i ngā āhuatanga ahotea, matekai rānei. Ngā āhuatanga moe 31,32,36,37, 38. I whakatauritehia e mātou te hanganga kua whakatūria i mua o te ribosome moe ki te mahere cryo-EM o te ribosome E. cuniculi. Mō te hononga, i whakamahia ngā ribosome S. cerevisiae i roto i te matatini me te āhuatanga moe Stm138, ngā ribosome mawhitiwhiti i roto i te matatini me te āhuatanga Lso232, me ngā ribosome V. necatrix i roto i te matatini me ngā āhuatanga Mdf1 me Mdf231. I taua wā anō, i kitea e mātou te kiato cryo-EM e rite ana ki te āhuatanga okiokinga Mdf1. He rite ki te herenga o Mdf1 ki te ribosome V. necatrix, ka herea hoki e Mdf1 ki te ribosome E. cuniculi, ka ārai i te pae E o te ribosome, ā, ka āwhina pea ki te wātea o ngā ribosome ina kore e mahi ngā pua pirinoa i te wā e whakakorengia ai te tinana (Pikitia 2).
Ka āraia e te Mdf1 te pae E o te ribosome, e āhua nei ka āwhina i te whakakore i te ribosome ina kore e mahi ngā spore pirinoa i roto i te pūnaha io. I roto i te hanganga o te ribosome E. cuniculi, i kitea e mātou ka hanga e te Mdf1 he hononga kāore i te mōhiotia i mua ki te take o te ribosome L1, te wāhanga o te ribosome e āwhina ana i te tukunga o te tRNA kua whakakorea te acylated mai i te ribosome i te wā e hanga pūmua ana. E kī ana ēnei hononga ka wehe te Mdf1 i te ribosome mā te whakamahi i te tikanga kotahi me te tRNA kua whakakorea te acylated, e whakarato ana i tētahi whakamārama pea mō te tango a te ribosome i te Mdf1 hei whakahohe anō i te hanga pūmua.
Heoi, i whakaatuhia e tā mātou hanganga tētahi hononga kāore i te mōhiotia i waenganui i a Mdf1 me te waewae ribosome L1 (te wāhanga o te ribosome e āwhina ana ki te tuku i te tRNA kua whakakorea te acylated mai i te ribosome i te wā hanga pūmua). Ina koa, ka whakamahia e Mdf1 ngā hononga ōrite ki te wāhanga tuke o te ngota tRNA kua whakakorea te acylated (Pikitia 2). I whakaatuhia e tēnei tauira ngota ngota kāore i te mōhiotia i mua ko te wehenga o Mdf1 mai i te ribosome mā te whakamahi i te tikanga ōrite ki te tRNA kua whakakorea te acylated, e whakamārama ana i te tango a te ribosome i tēnei tauwehe moe hei whakahohe anō i te hanga pūmua.
I te hanganga o te tauira rRNA, i kitea e mātou he kongakonga rRNA kua takai rerekē te ribosome E. cuniculi, i tapaina e mātou he rRNA kua hanumi (Pikitia 3). I roto i ngā ribosome e horapa ana i ngā rohe e toru o te ora, ka takai te rRNA ki roto i ngā hanganga e honoa ai te nuinga o ngā turanga rRNA tetahi ki tetahi, ka taunekeneke rānei ki ngā pūmua ribosomal38,39,40. Heoi, i roto i ngā ribosome E. cuniculi, te āhua nei ka takahia e ngā rRNA tēnei mātāpono takai mā te huri i ētahi o ā rātou helices ki ngā rohe rRNA kua tuwhera.
Te hanganga o te porowhita rRNA H18 25S i roto i te S. cerevisiae, V. necatrix, me te E. cuniculi. I roto i ngā ribosome e horapa ana i ngā rohe ora e toru, ka takai tēnei hononga ki roto i tētahi porowhita RNA kei roto he 24 ki te 34 toenga. I roto i te Microsporidia, he rerekē, ka whakaitihia haeretia tēnei hononga rRNA ki ngā hononga kotahi-aho e rua e whai kiko ana ki te uridine, e 12 noa iho ngā toenga. Ko te nuinga o ēnei toenga ka tukuna ki ngā whakarewa. E whakaatu ana te pikitia ko ngā microsporidia pirinoa te āhua nei kei te takahi i ngā mātāpono whānui o te takai rRNA, kei reira ngā turanga rRNA e honoa ana ki ētahi atu turanga, e uru ana rānei ki ngā taunekeneke rRNA-pūmua. I roto i te microsporidia, ka takai ētahi kongakonga rRNA ki tētahi takai kāore e pai, ka huri te porowhita rRNA o mua hei kongakonga kotahi-aho e toro ana tata tonu ki te rārangi tika. Mā te noho mai o ēnei rohe rerekē ka taea e te microsporidia rRNA te here i ngā kongakonga rRNA tawhiti mā te whakamahi i te iti rawa o ngā turanga RNA.
Ko te tauira tino kitea o tēnei whakawhiti whanaketanga ka kitea i roto i te H18 25S rRNA helix (Pikitia 3). I roto i ngā momo mai i te E. coli ki te tangata, kei roto i ngā turanga o tēnei rRNA helix he 24-32 ngā nucleotides, ka hanga he helix āhua koretake. I roto i ngā hanganga ribosomal kua tautuhia i mua mai i te V. necatrix me te P. locustae,31,32, kāore i te whiria ngā turanga o te H18 helix, engari ka tiakina te hononga turanga nucleotide. Heoi, i roto i te E. cuniculi, ka noho tēnei wāhanga rRNA hei hononga poto rawa 228UUUUGU232 me te 301UUUUUUUUUU307. Kāore i rite ki ngā wāhanga rRNA noa, kāore ēnei hononga whai uridine e whiria, e whakapā nui rānei ki ngā pūmua ribosomal. Engari, ka whakamahia e rātou he hanganga tuwhera-whakarewa me te tuwhera katoa, e toro tika ana ngā aho rRNA. Mā tēnei āhua totoro e whakamārama te whakamahinga a E. cuniculi i ngā turanga RNA 12 anake hei whakakī i te āputa 33 Å i waenganui i ngā porowhita rRNA H16 me H18, ko ētahi atu momo ia e hiahia ana kia rua te nui ake o ngā turanga rRNA hei whakakī i te āputa.
Nō reira, ka taea e tātou te whakaatu, mā te takai pūngao koretake, kua whakawhanakehia e ngā microsporidia pirinoa he rautaki hei whakawhāiti i aua wāhanga rRNA e tiakina whānuitia ana puta noa i ngā momo i roto i ngā rohe e toru o te ora. Te āhua nei, mā te kohikohi i ngā whakarerekētanga e huri ana i ngā poro rRNA hei hononga poly-U poto, ka taea e E. cuniculi te hanga i ngā kongakonga rRNA rerekē kei roto he iti noa ngā nucleotide hei here i ngā kongakonga rRNA tawhiti. Mā tēnei ka āwhina i te whakamārama me pēhea te whakatutuki a ngā microsporidia i tētahi whakahekenga nui i roto i tō rātou hanganga ngota taketake me te kore e ngaro tō rātou hanganga hanganga me te mahi.
Ko tētahi atu āhuatanga rerekē o te rRNA E. cuniculi ko te āhua o te rRNA me te kore he matotoru (Pikitia 4). Ko ngā pūkohu he nucleotides kāore he takirua turanga e miro ana i waho o te RNA helix, kaua e huna ki roto. Ko te nuinga o ngā pūkohu rRNA he piripiri ngota, e āwhina ana ki te here i ngā pūmua ribosomal tata, i ētahi atu maramara rRNA rānei. Ko ētahi o ngā pūkohu he hononga, e āhei ai te rRNA he piko me te takai pai mō te hanga pūmua whai hua 41.
a Kāore he putanga rRNA (te tau nama a S. cerevisiae) i roto i te hanganga ribosome E. cuniculi, engari kei roto i te nuinga o ngā eukaryote kē atu b E. coli, S. cerevisiae, H. sapiens, me E. cuniculi ribosome ā-roto. Kāore i roto i ngā pirinoa te nuinga o ngā pupuhi rRNA tawhito, tino tiakina. Mā ēnei matotorutanga ka whakau te hanganga ribosome; nō reira, ko tō rātou ngaronga i roto i te microsporidia e tohu ana i te iti o te pumau o te takai rRNA i roto i ngā pirinoa microsporidia. Ko te whakataurite ki ngā kakau P (ngā kakau L7/L12 i roto i ngā huakita) e whakaatu ana ko te ngaronga o ngā pupuhi rRNA i ētahi wā ka rite ki te putanga mai o ngā pupuhi hou i te taha o ngā pupuhi ngaro. He pupuhi tawhito te H42 helix i roto i te 23S/28S rRNA (U1206 i roto i te Saccharomyces cerevisiae) e kiia ana he 3.5 piriona tau te pakeke nā tōna tiakitanga i roto i ngā wāhanga e toru o te ora. I roto i te microsporidia, ka whakakorea tēnei pupuhi. Heoi anō, i puta mai tētahi pūkohu hou i te taha o te pūkohu i ngaro (A1306 i roto i te E. cuniculi).
He mea whakamiharo, i kitea e mātou kāore i roto i ngā ribosome E. cuniculi te nuinga o ngā pupuhi rRNA e kitea ana i roto i ētahi atu momo, tae atu ki te neke atu i te 30 ngā pupuhi e tiakina ana i roto i ētahi atu eukaryote (Pikitia 4a). Mā tēnei ngaronga ka whakakorea te maha o ngā hononga i waenga i ngā waeine ribosomal me ngā poro rRNA tata, i ētahi wā ka hangaia he āputa nui i roto i te ribosome, ka nui ake te pūngao o te ribosome E. cuniculi ki te whakataurite ki ngā ribosome tuku iho (Pikitia 4b). He mea nui, i kitea e mātou ko te nuinga o ēnei pupuhi i ngaro hoki i roto i ngā hanganga ribosome V. necatrix me P. locustae kua tautuhia i mua, i warewarehia e ngā tātaritanga hanganga o mua31,32.
I ētahi wā, ko te ngaronga o ngā pupuhi rRNA ka haere tahi me te whanaketanga o ngā pupuhi hou i te taha o te pupuhi ngaro. Hei tauira, kei roto i te kākano-P ribosomal he pupuhi U1208 (i roto i te Saccharomyces cerevisiae) i ora mai i te E. coli ki te tangata, nō reira e kiia ana he 3.5 piriona tau te pakeke. I te wā o te hanga pūmua, ka āwhina tēnei pupuhi i te kākano P ki te neke i waenga i ngā āhua tuwhera me ngā āhua kati kia taea ai e te ribosome te tiki i ngā āhuatanga whakamāori me te tuku i a rātou ki te pae hohe. I roto i ngā ribosome E. cuniculi, kāore tēnei matotorutanga; heoi, ko te matotorutanga hou (G883) kei roto i ngā takirua turanga e toru anake ka taea te whai wāhi ki te whakahoki mai i te ngāwari pai o te kākano P (Pikitia 4c).
E whakaatu ana ā mātou raraunga mō te rRNA kāore he pupuhi, kāore te whakaiti i te rRNA e herea noa ki te ngaronga o ngā huānga rRNA i runga i te mata o te ribosome, engari ka uru atu pea ki te karihi ribosome, ka puta he hapa ngota motuhake ki ngā pirinoa kāore anō kia whakaahuatia i roto i ngā pūtau ora kore utu.
I muri i te whakatauira i ngā pūmua ribosomal ā-pūtake me te rRNA, i kitea e mātou kāore e taea e ngā wāhanga ribosomal tikanga te whakamārama i ngā wāhanga e toru o te ahua cryo-EM. E rua o ēnei kongakonga he iti te rahi o ngā ngota (Pikitia 5, Pikitia Tāpiri 8). Ko te wāhanga tuatahi kei waenganui i ngā pūmua ribosomal uL15 me eL18 i te tūranga e nohoia ana e te C-terminus o eL18, e whakapotohia ana i roto i te E. cuniculi. Ahakoa kāore e taea e mātou te whakatau i te tuakiri o tēnei ngota, ko te rahi me te āhua o tēnei moutere kiato e whakamāramahia pai ana e te aroaro o ngā ngota spermidine. Ko tōna herenga ki te ribosome ka whakauhia e ngā whakarerekētanga motuhake ki te microsporidia i roto i ngā pūmua uL15 (Asp51 me Arg56), e ahua nei ka whakanui ake i te hononga o te ribosome ki tēnei ngota iti, nā te mea ka taea e uL15 te takai i te ngota iti ki roto i te hanganga ribosomal. Pikitia Tāpiri 2). 8, raraunga tāpiri 1, 2).
Whakaahua Cryo-EM e whakaatu ana i te aroaro o ngā nucleotide i waho o te ribose e herea ana ki te ribosome E. cuniculi. I roto i te ribosome E. cuniculi, kei te wāhi kotahi tēnei nucleotide me te nucleotide 25S rRNA A3186 (te tau nama Saccharomyces cerevisiae) i roto i te nuinga o ngā ribosome eukaryotic. b I roto i te hanganga ribosomal o E. cuniculi, kei waenganui i ngā pūmua ribosomal uL9 me eL20 tēnei nucleotide, ā, mā reira ka pumau te hononga i waenga i ngā pūmua e rua. cd tātaritanga tiaki raupapatanga eL20 i waenga i ngā momo microsporidia. Ko te rākau phylogenetic o ngā momo Microsporidia (c) me te whakaritenga raupapatanga maha o te pūmua eL20 (d) e whakaatu ana ko ngā toenga here-nucleotide F170 me K172 e tiakina ana i roto i te nuinga o ngā Microsporidia noa, haunga a S. lophii, haunga a Microsporidia peka wawe, i pupuri i te toronga rRNA ES39L. e E whakaatu ana tēnei pikitia kei roto noa i te eL20 o te ira microsporidia tino whakaitihia ngā toenga here-nucleotide F170 me K172, engari kāore i roto i ētahi atu eukaryote. I te nuinga, e kī ana ēnei raraunga kua whakawhanakehia e ngā ribosome Microsporidian he pae here nucleotide e āhua ana ki te here i ngā ngota AMP me te whakamahi i aua mea hei whakau i ngā taunekeneke pūmua-pūmua i roto i te hanganga ribosomal. Ko te tiakitanga teitei o tēnei pae here i roto i te Microsporidia me tōna ngaro i roto i ētahi atu eukaryote e kī ana ka whakaratohia e tēnei pae he painga oranga whiriwhiri mō te Microsporidia. Nō reira, ko te pute here-nucleotide i roto i te ribosome microsporidia ehara i te mea he āhuatanga pirau, he momo mutunga rānei o te whakahekenga rRNA e ai ki te whakaahuatanga o mua, engari he auaha whanaketanga whai hua e taea ai e te ribosome microsporidia te here tika i ngā ngota iti, me te whakamahi i a rātou hei poraka hanga ngota. poraka hanga mō ngā ribosome. Nā tēnei kitenga ka waiho te ribosome microsporidia hei ribosome anake e mōhiotia ana e whakamahi ana i te nucleotide kotahi hei poraka hanga hanganga. f Ara whanaketanga whakapae i ahu mai i te here nucleotide.
Kei te hononga i waenga i ngā pūmua ribosomal uL9 me eL30 te tuarua o ngā matūriki iti te taumaha (Pikitia 5a). I whakaahuahia tēnei hononga i mua i roto i te hanganga o te ribosome Saccharomyces cerevisiae hei pae here mō te nucleotide 25S o te rRNA A3186 (he wāhanga o te toronga rRNA ES39L)38. I whakaaturia i roto i ngā ribosome P. locustae ES39L kua pirau, ka herea e tēnei hononga he nucleotide kotahi kāore i te mōhiotia 31, ā, e whakaarohia ana ko tēnei nucleotide he momo whakamutunga kua whakaitihia o te rRNA, ko te roa o te rRNA he ~130-230 turanga. Ka whakaitihia te ES39L ki te nucleotide kotahi 32.43. E tautoko ana ā mātou whakaahua cryo-EM i te whakaaro ka taea te whakamārama i te matotoru e ngā nucleotide. Heoi, ko te whakataunga teitei ake o tā mātou hanganga i whakaatu ko tēnei nucleotide he ngota extraribosomal, tera pea ko te AMP (Pikitia 5a, b).
Kātahi mātou ka pātai mēnā i puta te pae here nucleotide i roto i te ribosome E. cuniculi, mēnā rānei i mua. Nā te mea ko ngā toenga Phe170 me Lys172 i roto i te pūmua ribosomal eL30 te nuinga o te wā e ārahi ana i te here nucleotide, i aromatawaihia e mātou te tiakitanga o ēnei toenga i roto i ngā eukaryote e 4396. Pērā i te take o te uL15 i runga ake nei, i kitea e mātou ko ngā toenga Phe170 me Lys172 he tino tiakina i roto i ngā Microsporidia noa iho, engari kāore i roto i ētahi atu eukaryote, tae atu ki te Microsporidia Mitosporidium me Amphiamblys kāore i te tino kitea, kāore hoki i whakaitihia te wāhanga rRNA ES39L 44, 45, 46 (Pikitia 5c). -e).
Ina huihuia, e tautoko ana ēnei raraunga i te whakaaro kua whanakehia e E. cuniculi me ētahi atu microsporidia matua te kaha ki te hopu pai i te maha o ngā matū iti i roto i te hanganga ribosome hei utu mō te hekenga o ngā taumata rRNA me te pūmua. I roto i te mahi pera, kua whakawhanakehia e rātou he kaha ahurei ki te here i ngā nucleotide i waho o te ribosome, e whakaatu ana ka utua e ngā hanganga ngota pirinoa mā te hopu i te nui o ngā matū iti me te whakamahi i a rātou hei tauira hanganga o ngā kongakonga RNA me te pūmua kua pakaru.
Ko te tuatoru o ngā wāhanga kāore i whakaatahia o tā mātou mahere cryo-EM, i kitea i roto i te wāhanga ribosomal nui. Ko te taumira teitei (2.6 Å) o tā mātou mahere e kī ana nō ngā pūmua tēnei kiato me ngā huinga ahurei o ngā toenga mekameka taha nui, i āhei ai mātou ki te tautuhi i tēnei kiato hei pūmua ribosomal kāore i te mōhiotia i mua i tautuhia e mātou. I tapaina ko msL2 (pūmua motuhake ki te Microsporidia L2) (tikanga, pikitia 6). I whakaatuhia e tā mātou rapunga homology kei te tiakina te msL2 i roto i te rōpū Microsporidia o te puninga Encephaliter me Orosporidium, engari kāore i roto i ētahi atu momo, tae atu ki ētahi atu Microsporidia. I roto i te hanganga ribosomal, kei roto i te āputa i hangaia e te ngaronga o te rRNA ES31L kua roa. I roto i tēnei āputa, ka āwhina a msL2 ki te whakau i te takai rRNA, ā, ka taea te utu mō te ngaronga o te ES31L (Pikitia 6).
a Te kiato irahiko me te tauira o te pūmua ribosomal motuhake-Microsporidia msL2 i kitea i roto i ngā ribosome E. cuniculi. b Ko te nuinga o ngā ribosome eukaryotic, tae atu ki te ribosome 80S o Saccharomyces cerevisiae, kua ngaro te whakanui ES19L rRNA i roto i te nuinga o ngā momo Microsporidian. Ko te hanganga o mua o te ribosome microsporidia V. necatrix e kī ana ko te ngaronga o te ES19L i roto i ēnei pirinoa ka utua e te whanaketanga o te pūmua ribosomal msL1 hou. I roto i tēnei rangahau, i kitea e mātou i whakawhanakehia hoki e te ribosome E. cuniculi tētahi atu pūmua RNA ribosomal hei utu mō te ngaronga o te ES19L. Heoi, he rerekē te hanganga me te whanaketanga o te takenga mai o te msL2 (i tēnei wā ko te pūmua ECU06_1135 whakapae) me te msL1. c Ko tēnei kitenga o te whakaputanga o ngā pūmua ribosomal msL1 me msL2 kāore i te hono ki te whanaketanga e kī ana mēnā ka kohia e ngā ribosome ngā whakarerekētanga kino i roto i ā rātou rRNA, ka taea e rātou te whakatutuki i ngā taumata rerekētanga o te hanganga i roto i tētahi wāhanga iti o ngā momo e pā tata ana. Tērā pea ka āwhina tēnei kitenga ki te whakamārama i te takenga mai me te whanaketanga o te ribosome mitochondrial, e mōhiotia ana mō tōna rRNA tino iti me te rerekētanga rerekē o te hanganga pūmua puta noa i ngā momo.
I whakaritea e mātou te pūmua msL2 ki te pūmua msL1 kua whakaahuatia i mua, koia anake te pūmua ribosomal motuhake-microsporidia e mōhiotia ana i roto i te ribosome V. necatrix. I hiahia mātou ki te whakamātautau mēnā he whanaungatanga whanaketanga a msL1 me msL2. I whakaatuhia e tā mātou tātaritanga kei roto i te kōhao kotahi a msL1 me msL2 i roto i te hanganga ribosomal, engari he rerekē ngā hanganga tuatahi me te tuatoru, e tohu ana i tō rātou takenga whanaketanga motuhake (Pikitia 6). Nō reira, ko tā mātou kitenga o msL2 e whakaatu ana i ngā taunakitanga ka taea e ngā rōpū o ngā momo eukaryotic kiato te whakawhanake motuhake i ngā pūmua ribosomal motuhake hei utu mō te ngaronga o ngā kongakonga rRNA. He mea whakamiharo tēnei kitenga nā te mea kei roto i te nuinga o ngā ribosome eukaryotic cytoplasmic he pūmua pumau, tae atu ki te whānau kotahi o ngā pūmua ribosomal 81. Ko te putanga mai o msL1 me msL2 i roto i ngā momo microsporidia hei whakautu ki te ngaronga o ngā wāhanga rRNA kua roa e kī ana ko te whakahekenga o te hanganga ngota o te pirinoa ka meinga ngā pirinoa ki te rapu i ngā whakarerekētanga utu, ka mutu ka arahi pea ki te whiwhinga i roto i ngā taupori pirinoa rerekē.
Hei whakamutunga, i te otinga o tā mātou tauira, i whakaritea e mātou te hanganga o te ribosome E. cuniculi ki te mea i matapaetia mai i te raupapatanga ira. He maha ngā pūmua ribosomal, tae atu ki te eL14, eL38, eL41, me te eS30, i whakaarohia i mua kua ngaro i te ira E. cuniculi nā te ngaronga o ō rātou hoa i te ira E. cuniculi. E matapaetia ana hoki te ngaronga o te maha o ngā pūmua ribosomal i roto i te nuinga o ngā pirinoa intracellular me ngā endosymbionts kua tino whakaitihia. Hei tauira, ahakoa kei roto i te nuinga o ngā huakita ora kore utu te whānau kotahi o ngā pūmua ribosomal 54, 11 noa iho o ēnei whānau pūmua he hoa ka kitea i roto i ia ira kua tātarihia o ngā huakita kua herea e te manaaki. Hei tautoko i tēnei whakaaro, kua kitea te ngaronga o ngā pūmua ribosomal i roto i te V. necatrix me te P. locustae microsporidia, kāore nei i te whai pūmua eL38 me eL4131,32.
Heoi, e whakaatu ana ā mātou hanganga ko eL38, eL41, me eS30 anake ka ngaro i roto i te ribosome E. cuniculi. I tiakina te pūmua eL14, ā, i whakaatuhia e tā mātou hanganga he aha i kore ai e kitea tēnei pūmua i roto i te rapunga homology (Pikitia 7). I roto i ngā ribosome E. cuniculi, ka ngaro te nuinga o te pae here eL14 nā te whakahekenga o te ES39L kua whakanuia e te rRNA. I te ngaro o te ES39L, ka ngaro te nuinga o te hanganga tuarua o eL14, ā, 18% noa iho o te raupapa eL14 i rite i roto i te E. cuniculi me te S. cerevisiae. He mea whakamiharo tēnei tiaki raupapa ngoikore nā te mea ko Saccharomyces cerevisiae me te Homo sapiens—ngā rauropi i whanake 1.5 piriona tau te tawhiti—he nui atu i te 51% o ngā toenga ōrite i roto i te eL14. Nā tēnei ngaronga rerekē o te tiakitanga i whakamārama he aha i tāpirihia ai a E. cuniculi eL14 hei pūmua M970_061160 pea, kaua hei pūmua ribosomal eL1427.
ā, i ngaro i te ribosome Microsporidia te toronga rRNA ES39L, i whakakorea ai te pae here pūmua ribosomal eL14. I te ngaronga o te ES39L, ka ngaro te hanganga tuarua o te pūmua microspore eL14, ka pirau te α-helix here-rRNA o mua hei porowhita roa iti. b E whakaatu ana te whakaritenga raupapa maha he tino tiakina te pūmua eL14 i roto i ngā momo eukaryotic (57% te ōritetanga raupapa i waenga i te rewena me ngā homologue tangata), engari he iti te tiaki me te rerekē i roto i te microsporidia (e kore e neke atu i te 24% o ngā toenga he ōrite ki te homologue eL14). mai i S. cerevisiae, H. sapiens rānei). Ko tēnei tiaki raupapa ngoikore me te rerekētanga hanganga tuarua e whakamārama ana he aha i kore ai te homologue eL14 i kitea i roto i te E. cuniculi me te take i whakaarohia ai kua ngaro tēnei pūmua i roto i te E. cuniculi. He rerekē, i mua i whakarārangihia a E. cuniculi eL14 hei pūmua M970_061160 pea. E kī ana tēnei kitenga he nui rawa te whakatau tata ki te kanorau o te ira microsporidia: ko ētahi ira e whakaarohia ana kua ngaro i roto i ngā microsporidia kei te tiakina tonutia, ahakoa i roto i ngā āhua tino rerekē; engari, e whakaarohia ana ko ētahi hei waehere mō ngā ira microsporidia mō ngā pūmua motuhake ki te kutukutu (hei tauira, ko te pūmua whakapae M970_061160) e waehere ana mō ngā pūmua tino kanorau e kitea ana i roto i ētahi atu eukaryote.
E whakaatu ana tēnei kitenga ka taea e te whakakorenga o te rRNA te arahi ki te ngaronga nui o te tiakitanga raupapa i roto i ngā pūmua ribosomal tata, ka kore ai e kitea ēnei pūmua mō ngā rapunga homology. Nō reira, ka taea e tātou te whakanui rawa i te nui o te whakahekenga ngota i roto i ngā rauropi ira iti, nā te mea ko ētahi pūmua e whakaarohia ana kua ngaro kei te mau tonu, ahakoa kei roto i ngā āhua kua tino rerekē.
Me pēhea e taea ai e ngā pirinoa te pupuri i te mahi a ā rātou mīhini ngota i raro i ngā āhuatanga o te whakaiti nui o te ira? Ka whakautua e tā mātou rangahau tēnei pātai mā te whakaahua i te hanganga ngota matatini (ribosome) o E. cuniculi, he rauropi me tētahi o ngā ira eukaryotic iti rawa.
E rua tekau tau te roa o te mōhiotia ana ko ngā ngota pūmua me ngā ngota RNA i roto i ngā pirinoa moroiti he rerekē i ō rātou ngota ōrite i roto i ngā momo ora-kore nā te mea kāore i a rātou ngā pokapū whakahaere kounga, ka whakaitihia ki te 50% o tō rātou rahi i roto i ngā moroiti ora-kore, me ētahi atu. he maha ngā whakarerekētanga ngoikore e whakararu ana i te takai me te mahi. Hei tauira, ko ngā ribosome o ngā rauropi ira iti, tae atu ki te maha o ngā pirinoa ā-roto me ngā endosymbionts, e tika ana kia kore he maha o ngā pūmua ribosomal me te kotahi hautoru o ngā nucleotide rRNA ki te whakataurite ki ngā momo ora-kore 27, 29, 30, 49. Heoi, ko te āhua o te mahi a ēnei ngota i roto i te pirinoa he mea ngaro tonu, e rangahauhia ana mā te whakataurite i ngā ira.
E whakaatu ana tā mātou rangahau ka taea e te hanganga o ngā macromolecules te whakaatu i ngā āhuatanga maha o te whanaketanga e uaua ana ki te tango mai i ngā rangahau ira whakataurite tuku iho o ngā pirinoa ā-roto me ētahi atu rauropi e herea ana e te manaaki (Pikitia Tāpiri 7). Hei tauira, ko te tauira o te pūmua eL14 e whakaatu ana ka taea e tātou te whakanui rawa i te nui o te whakahekenga o te taputapu ngota i roto i ngā momo pirinoa. E whakaponohia ana ināianei he rau ngā ira motuhake ki ngā pirinoa encephalitic. Heoi, e whakaatu ana ā mātou hua ko ētahi o ēnei ira āhua motuhake he momo rerekē noa iho o ngā ira e kitea ana i roto i ētahi atu eukaryotes. Heoi anō, ko te tauira o te pūmua msL2 e whakaatu ana me pēhea tātou e wareware ana ki ngā pūmua ribosomal hou me te whakaiti i te ihirangi o ngā mīhini ngota pirinoa. Ko te tauira o ngā ngota iti e whakaatu ana me pēhea tātou e wareware ai ki ngā auahatanga tino auaha i roto i ngā hanganga ngota pirinoa ka taea te hoatu ki a rātou he mahi koiora hou.
Ina huihuia ēnei hua, ka whakapai ake i tō tātou māramatanga ki ngā rerekētanga i waenga i ngā hanganga ngota o ngā rauropi e herea ana e te manaaki me ō rātou hoa i roto i ngā rauropi ora kore utu. E whakaatu ana mātou ko ngā mīhini ngota, kua roa e whakaarohia ana he whakaiti, he pirau, ā, ka pāngia e ngā whakarerekētanga ngoikore, engari he huinga āhuatanga hanganga rerekē kua warewarehia.
I tētahi atu taha, ko ngā maramara rRNA kore-pūmau me ngā maramara honohono i kitea e mātou i roto i ngā ribosome o E. cuniculi e kī ana ka taea e te whakaiti ira te whakarerekē i ngā wāhanga o te mīhini ngota taketake e tiakina ana i roto i ngā rohe e toru o te ora – i muri i te tata ki te 3.5 piriona tau. te whanaketanga motuhake o ngā momo.
He mea tino hira ngā maramara rRNA kore-puku me te whakakotahi i roto i ngā ribosome E. cuniculi i runga i ngā rangahau o mua mō ngā ngota RNA i roto i ngā huakita endosymbiotic. Hei tauira, i roto i te aphid endosymbiont Buchnera aphidicola, kua whakaatuhia he hanganga tairongo-mahana ngā ngota rRNA me te tRNA nā te mea he rerekē te hanganga A+T me te nui o ngā takirua turanga kore-canonical20,50. Ko ēnei huringa i roto i te RNA, me ngā huringa i roto i ngā ngota pūmua, e whakaarohia ana inaianei ko te take o te whakawhirinaki nui o ngā endosymbionts ki ngā hoa me te kore e taea e ngā endosymbionts te whakawhiti wera 21, 23. Ahakoa he rerekē te hanganga o te rRNA microsporidia parasitic, ko te āhua o ēnei huringa e kī ana ko te iti o te pumau wera me te whakawhirinaki nui ake ki ngā pūmua chaperone he āhuatanga noa pea o ngā ngota RNA i roto i ngā rauropi he iti ake ngā ira.
I tētahi atu taha, e whakaatu ana ā mātou hanganga kua whanakehia e ngā microsporidia pirinoa he kaha ahurei ki te ātete i ngā maramara rRNA me ngā pūmua kua tiakina whānuitia, e whakawhanake ana i te kaha ki te whakamahi i ngā metabolites iti e nui ana, e wātea ana hoki hei tauira hanganga o ngā maramara rRNA me ngā pūmua kua pirau. Te whakahekenga hanganga ngota. . E tautokona ana tēnei whakaaro e te meka ko ngā ngota iti e whakakapi ana i te ngaronga o ngā maramara pūmua i roto i te rRNA me ngā ribosome o E. cuniculi ka herea ki ngā toenga motuhake ki te microsporidia i roto i ngā pūmua uL15 me eL30. E kī ana tēnei ko te herenga o ngā ngota iti ki ngā ribosome he hua pea o te kōwhiringa pai, i reira kua tīpakohia ngā whakarerekētanga motuhake ki te Microsporidia i roto i ngā pūmua ribosomal mō tō rātou kaha ki te whakanui ake i te hononga o ngā ribosome ki ngā ngota iti, ka arahi pea ki ngā rauropi ribosomal whai hua ake. E whakaatu ana te kitenga i tētahi auahatanga atamai i roto i te hanganga ngota o ngā pirinoa moroiti, ā, e homai ana ki a tātou he māramatanga pai ake mō te pupuri a ngā hanganga ngota pirinoa i tā rātou mahi ahakoa te whanaketanga whakaiti.
I tēnei wā, kāore anō kia mārama te tautuhi i ēnei ngota iti. Kāore i te mārama he aha te rerekētanga o te āhua o ēnei ngota iti i roto i te hanganga ribosomal i waenga i ngā momo microsporidia. Ina koa, kāore i te mārama he aha te herenga nucleotide i kitea ai i roto i ngā ribosome o E. cuniculi me P. locustae, kaua i roto i ngā ribosome o V. necatrix, ahakoa te noho o te toenga F170 i roto i ngā pūmua eL20 me K172 o V. necatrix. Tērā pea nā te toenga 43 uL6 (kei te taha o te pute herenga nucleotide) tēnei mukunga, arā, ko te tyrosine i roto i te V. necatrix, ehara i te threonine i roto i te E. cuniculi me P. locustae. Ka taea e te mekameka taha kakara matotoru o Tyr43 te whakararuraru i te herenga nucleotide nā te taupatupatu steric. Waihoki, ko te mukunga nucleotide e kitea ana, tērā pea nā te iti o te taumira o te whakaahua cryo-EM, e whakararu ana i te whakatauira o ngā kongakonga ribosomal o V. necatrix.
I tētahi atu taha, e kī ana tā mātou mahi he kaha auaha pea te tukanga pirau o te ira. Ina koa, ko te hanganga o te ribosome E. cuniculi e kī ana ko te ngaronga o te rRNA me ngā kongakonga pūmua i roto i te ribosome microsporidia ka puta he pēhanga whanaketanga e whakatairanga ana i ngā huringa o te hanganga ribosome. Ka puta mai ēnei momo rerekētanga i tawhiti atu i te pae hohe o te ribosome, ā, te āhua nei ka āwhina ki te pupuri (ki te whakaora rānei) i te huihuinga ribosome tino pai ka whakararuraruhia e te rRNA kua whakaitihia. E kī ana tēnei ko te auahatanga nui o te ribosome microsporidia te āhua nei kua whanake hei hiahia ki te ārai i te nekenekehanga ira.
Tērā pea ka tino kitea tēnei mā te herenga nucleotide, kāore anō kia kitea i roto i ētahi atu rauropi tae noa mai ki tēnei wā. Ko te meka kei roto ngā toenga herenga nucleotide i roto i ngā microsporidia noa, engari kāore i roto i ētahi atu eukaryote, e kī ana ehara ngā pae herenga nucleotide i te mea he toenga noa iho e tatari ana kia ngaro, i te pae whakamutunga rānei mō te rRNA kia whakahokia ki te āhua o ngā nucleotide takitahi. Engari, te āhua nei he āhuatanga whai hua tēnei pae i whanake pea i roto i ētahi rauna o te kōwhiringa pai. Ko ngā pae herenga nucleotide he hua taha pea nō te kōwhiringa taiao: ina pakaru te ES39L, ka akiakihia ngā microsporidia ki te rapu utu hei whakahoki mai i te biogenesis ribosome tino pai i te ngaro o te ES39L. I te mea ka taea e tēnei nucleotide te whai i ngā hononga ngota o te nucleotide A3186 i roto i te ES39L, ka noho te ngota nucleotide hei poraka hanga o te ribosome, ka pai ake te herenga mā te whakarerekētanga o te raupapa eL30.
Mō te whanaketanga ngota o ngā pirinoa ā-roto, e whakaatu ana tā mātou rangahau kāore ngā kaha o te kōwhiringa taiao a Darwin me te hekenga ira o te pirau ira e mahi tahi ana, engari e hurihuri ana. Tuatahi, ka whakakorea e te hekenga ira ngā āhuatanga nui o ngā koiora koiora, ka tino hiahiatia he utu. Mēnā ka ea i ngā pirinoa tēnei hiahia mā te kōwhiringa taiao a Darwin, ka whai wāhi ā rātou macromolecules ki te whakawhanake i ō rātou āhuatanga tino whakamiharo me te auaha. He mea nui, ko te whanaketanga o ngā pae here nucleotide i roto i te ribosome E. cuniculi e kī ana ko tēnei tauira ngaronga-ki-te-whiwhi o te whanaketanga ngota ehara i te mea ka whakakore noa i ngā whakarerekētanga kino, engari i ētahi wā ka whakawhiwhia he mahi hou ki ngā macromolecules pirinoa.
He rite tonu tēnei whakaaro ki te ariā taurite neke a Sewell Wright, e kī ana ko te pūnaha tino kaha o te kōwhiringa taiao ka whakawhāiti i te kaha o ngā rauropi ki te auaha51,52,53. Heoi, ki te whakararuraru te hekenga ira i te kōwhiringa taiao, ka taea e ēnei hekenga te whakaputa i ngā huringa kāore i te urutau (e kino ana rānei) engari ka arahi ki ētahi atu huringa e whakarato ana i te hauora teitei ake, i te mahi koiora hou rānei. E tautoko ana tā mātou anga i tēnei whakaaro mā te whakaatu ko te momo whakarerekētanga kotahi e whakaiti ana i te piko me te mahi o te koiora koiora te mea nui hei whakaoho i tōna whakapainga. E ai ki te tauira whanaketanga toa-toa, e whakaatu ana tā mātou rangahau ko te pirau o te ira, e kiia ana he tukanga whakaheke, he mea nui hoki hei akiaki i te auahatanga, i ētahi wā, ā, he maha pea ka taea e ngā macromolecules te whakamahi i aua mea.