3.2 Escherichia coli konstrueeritud bakteritüvede MG1655 (∆rluC ∆rlmE), MG1655
3.2.3 Konstrueeritud bakteritüvede MG1655 (∆rluC ∆rlmE), MG1655 (∆rluC
∆rluF), MG1655 (∆rluC ∆rlmKL ∆rlmN ∆rlmM) ribosoomide analüüs
Valitud tüvede (MG1655 (∆rluC ∆rlmE::Km), MG1655 (∆rluC ∆rlmKL ∆rluF), MG1655 (∆rluC ∆rlmKL ∆rlmN ∆rlmM)) ribosoomide ja subühikute analüüsimiseks fraktsioneeriti ribosoomid 15% - 40% sahharoosigradiendil ning visualiseeriti vastavalt metoodikas kirjeldatud meetoditele (ptk 2.9). Referentstüvedena kasutati tüvesid MG1655 wt, MG1655 (ΔrluC) ja JW3146-2 (∆rlmE::Km).
Joonisel 18 on näha, et konstrueeritud tüvedest (∆rluC ∆rlmKL ∆rluF ja ∆rluC ∆rlmKL
∆rlmN ∆rlmM) 70S, 50S ja 30S partiklite osakaal sarnaneb MG1655 referentstüvele. Samas on kõigil konstrueeritud tüvedel vähenenud polüsoomide osakaal võrreldes wt tüvega, mis viitab, et mRNA transleerimisel võib ribosoomidel kerge probleem olla.
∆rluC ∆rlmE mutandil (joonis 18) on selgelt eristuv sahharoosi gradiendi profiil: 70S
partiklite osakaal on oluliselt vähenenud 50S ja 30S partiklite osakaalud aga kasvanud. Lisaks on muutunud ka polüsoomide pilt. Võrreldes metsiktüüpi tüvega on ti- ja trisoome
silmnähtavalt vähem. Põhjenduseks võib pidada suurt vabade subühikute osakaalu ja
funktsionaalsete 70S ribosoomide vähest hulka, mis võib tuleneda probleemidest subühikute assambleerumisel. Selline sahharoosigradiendi profiil põhjendab ära ka oluliselt pikema lag-faasi ning aeglasema kasvukiiruse eksponentsiaalses lag-faasis (joonis 18). ∆rluC ja ∆rlmE tüved on küllaltki sarnased MG1655 wt tüvele, kuid ∆rlmE tüve puhul on näha veidi suurenenud vabade subühikute hulk. Bügl et al. poolt tehtud sahharoosi gradiendi profiil (joonis 17)
∆rlmE tüvest (ftsJ -) näitab ∆rluC ∆rlmE mutandile sarnasemat partiklite jaotumist, kus
45
vabade 50S ja 30S subühikute osakaal on suurenenud ning 70S ribosoomide osakaal
vähenenud (Bügl et al., 2000). Üks põhjus, miks antud bakalaureuse töö käigus saadud ∆rlmE tüve sahharoosi gradiendi profiil on teistsugune võib olla põhjustatud kompenseerivate mutatsioonide tekkest. Tan et al. on näidanud, et väikeste GTPaaside Obg ja EngA
üleekspressioon taastab ∆rlmE mutanttüvel wild type fenotüübi ja ribosoomide sahharoosi gradiendi profiili (Tan et al., 2002). Väljaselgitamaks, kas ∆rluC ∆rlmE mutandi fenotüüp on põhjustatud ainult rlmE geeni deletsioonist või süveneb kahe geeni puudumise koosmõjul või on seotud retsipienttüvega oleks vaja teha täiendavaid katseid, kus transduktsiooni
geeniülekandes oleks doonortüveks MG1665 ∆rluC::Km ning retsipienttüveks MG1655 (∆rlmE).
Joonis 17. Polüsoomide profiil 10% - 50% sahharoosi gradiendis. Võrreldud on HB24 (wt) ja HB23 (ftsJΔ567).
HB23 tüvest on deleteeritud ftsJ ehk rlmE geen. (Bügl et al., 2000).
46
Joonis 18. Ribosoomide fraktsioneerimine 15% - 40% sahsahharoosi gradiendis. Gradiendi kontsentratsioon muutub lineaarselt. Võrreldud on tüvede MG1655 wt, MG1655 (ΔrluC), JW3146-2, MG1655 (ΔrluC ΔrlmE::Km) MG1655 (∆rluC ∆rlmKL ∆rluF) ja MG1655 (∆rluC ∆rlmKL ∆rlmN ∆rlmM) fraktsioone.
47
Antud töös jäid vaatluse alt välja modifikatsiooniensüümid RluB, RluE ja RlmB, mis modifitseerivad nukleotiide vastavalt positsioonides U2605, U2457 ja G2251. Nendest modifikatsioonidest paigutuvad Green ja Nolleri poolt määratud 80 nukleotiidsesse piirkonda vaid RluE poolt katalüüsitav pseudouridiin Ψ2457. Samas modifikatsioon Gm2251 paikneb strateegiliselt olulises piirkonnas – P-lingus, mis on oluline peptidüül-tRNA seondumiseks.
Bakalaureusetöös saadud tulemustest võib järeldada, et kuigi enamus 23S rRNA domeeni V modifikatsioone ei pruugi olla Escherichia coli jaoks hädavajalikud, siis võib leiduda modifikatsioonide kombinatsioone, mille puudumise korral ei ole võimalik konstrueerida E.
coli tüve. Selle kindlamaks tõestamiseks on tulevikus vaja konstrueerida E. coli tüvesid, milles puuduvad erinevates kombinatsioonides domeeni V modifikatsioonid. Lisaks
võimalikule modifikatsioonide tähtsusele valgusünteesis võivad 23S rRNA modifikatsioonid olla seotud ka bakterirakkude omandatud kaitsemehhanismiga antibiootikumide vastu.
48 KOKKUVÕTE
Käesoleva töö eesmärk oli konstrueerida Escherichia coli tüvi, milles puuduvad kõik 23S rRNA domeeni V modifikatsioonid. Bakalaureuse töö käigus konstrueeriti Escherichia coli mutanttüvesid, kus puuduvad erinevates kombinatsioonides 23S rRNA domeeni V
modifikatsioonid. Konstrueeritud tüvede fenotüüpide iseloomustamiseks uuriti bakterirakkude temperatuuritundlikkust, kasvuparameetreid ning analüüsiti ribosomaalset koostist sahharoosi gradiendis fraktsioneerimisega.
Üksikute modifikatsioonide puudumine domeenis V ei mõjuta ribosoomide funktsionaalsust ja bakterirakkude fenotüüpi. Siiski arvatakse, et võib leiduda kombinatsioone domeenis V asuvatest modifikatsioonidest, mille puudumisel ei ole ribosoom funktsionaalne. Sellise tüve loomisel võib eeldada, et bakterirakkude ribosoomid on väga haiged või rakud pole
elujõulised. Antud töös jõuti sellise tüve loomisele lähemale. Modifikatsioonide olulisuse paremaks mõistmiseks ribosoomi funktsionaalsusele, oleks oluline jätkata Escherichia coli mutanttüvede loomisega, millest võimaluse korral puuduksid kõik domeeni V
modifikatsioonid.
Töös tehtud analüüsi käigus leiti kombinatsioon modifikatsioonidest, mille puudumine ei avalda mõju rakkude kasvukiirusele ega temperatuuritundlikkusele. Samas konstrueeriti ka tüvi, millel esines tugev kasvudefekt, oli suurenenud vabade subühikute osakaal ning 70S ribosoomide hulk oli oluliselt vähenenud.
Esialgsete tulemuste põhjal võib arvata, et tõenäoliselt leidub kombinatsioon 23S rRNA domeeni V modifikatsioonidest, mille puhul ribosoomid on väga defektsed. Kui sellise tüve loomine on võimalik, siis suure tõenäosusega on sellel tüvel väga tugev kasvudefekt.
49
On the importance of modifications in Eschericia coli 23S rRNA domain V
Triin Truu SUMMARY
Escherichia coli is one of the most used organisms for studying ribosomes and protein
synthesis. In all domains of life ribosomal RNA contains modifications. 23S rRNA contains a number of modifications around domain V peptidyl transferase center (PTC). It has been noted that the absence of single modifications around that area has almost no effect on ribosomal maturation, function or cell growth (Del Campo et al., 2001; Lövgren and Wikström, 2001; Conrad et al., 1998). Only the absence of methyltransferase/heat-shock protein RlmE/FtsJ thus the absence of methylation of U2552 seems to be somewhat important for ribosomal assembly. Deletion of this enzyme leads to decrease in 70S ribosomes and increase of free subunits which explains defects in growth rate (Bügl et al., 2000).
The aim of this thesis was to construct an Escherichia coli strain that lacked all of the
modifications located in the domain V of 23S rRNA. The experimenter was able to construct strains of E. coli lacking combinations of different domain V modifications. Some
combinations have almost no effect on ribosomal profile in sucrose gradient, cell growth and temperature sensitivity while a strain lacking two modification enzymes rluC and rlmE showed severe growth defects and decrease in 70S ribosomes. Interestingly the constructed strain showed increased sensitivity to lower temperatures whereas the methyltransferase absent from the strain is also a heat shock protein. All of the strains constructed showed decrease in polysome profile.
Even though most modifications in the PTC area may not be essential for ribosome assembly they could contribute to bacterial defense mechanism against various antibiotics targeting PTC. In order to investigate further the importance of modifications in 23S rRNA domain V the experimenter needs to construct more strains variating combinations of deleted
modification enzyme genes.
50 TÄNUSÕNAD
Autor tänab molekulaarbioloogia ja biokeemia labori meeldivat seltskonda. Eriline tänu kuulub juhendajale Aivar Liivile, professor Jaanus Remmele ja Margus Leppikule kannatlikkuse ja väärtuslike nõuannete eest.
51 KASUTATUD KIRJANDUS
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