Studies on the role of helix 69 of 23S rRNA in the factor-dependent stages of translation initiation, elongation, and termination
Date
2011-04-12
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Abstract
Käesoleva doktoritöö eesmärgiks oli selgitada i) ribosomaalse 23S rRNA heeliksi 69 võimalikku rolli valgusünteesi faktorkatalüüsitud etappides ning ii) heeliksis 69 sisalduvate pseudouridiinide rolli translatsiooni terminatsioonil. Lisaks on töös selgitatud ka pseudouridiini süntaasi RluD substraadispetsiifilisust määravaid tegureid.
Doktoritöö alguseks oli kogunenud märkimisväärne hulk struktuuripõhiseid viiteid heeliksi 69 osaluses kohta erinevates ribosoomi töötsükli osades. Käesoleva doktoritöö eesmärgiks oligi nende struktuuripõhiste hüpoteeside biokeemiline kontrollimine.
Töö eksperimentaalses osas asendati suunatud mutageneesi abil heeliksi 69 valitud positsioonides olevad natiivsed lämmastikalused teiste lämmastikalustega. Variantsed ribosoomid ekspresseeritid E.colis, affiinsuspuhastati ning nende ribosoomide aktiivsust analüüsiti erinevates in vitro katsesüsteemides. Katsetest saadud tulemused osutavad, et 23S rRNA heeliks 69 osaleb initsiatsioonifaktorite aktiivsuse regulatsioonis 70S initsiatsioonikompleksi moodustumisel ning et heeliks 69 on oluline ribosoomi üleminekul initsiatsioonifaasist protsessiivse elongatsiooni faasi. Pseudouridiinide eemaldamine heeliksist 69 pärssis peptiidi vabastamist polüpeptiidi vabanemisfaktori RF2 poolt, kuid ei mõjutanud RF2-ga struktuurilt väga sarnase vabanemisfaktori RF1 aktiivsust. See tulemus osutab erinevustele RF1 ja RF2 katalüüsimehhanismis. Lisaks osutavad meie tulemused, et pseudouridiini süntaasi RluD spetsiifikat heeliksi 69 suhtes suunab heeliksi 69 positsioonis 1916 asuv nukleotiid.
The aim of the present dissertation was to analyze i) the role of helix 69 of the ribosomal 23S rRNA in the the factor-catalyzed steps of protein synthesis and ii) the role of pseudouridines in helix 69 for translation termination. The factors determining the substrate specificity of the pseudouridine synthase RluD were also investigated. The involvement of helix 69 in ribosomal functioning has been suggested by x-ray and cryo-EM analyses of various ribosomal complexes. However, at the beginning of the present work the majority of the structure-based suggestions about the regulatory role of helix 69 had not been biochemically tested. In our studies, selected residues of helix 69 were replaced by site-directed mutagenesis, the variant ribosomes were expressed in E.coli in the presence of WT ribosomes and affinity-purified and tested for their activity in different in vitro assays. Our results indicate that helix 69 participates in the regulation of the activity of the initiation factors during the 70S initiation complex formation and is important for the transition of the ribosome from initiation to processive elongation. Depletion of pseudouridines from helix 69 impaired peptide release by release factor RF2 but not by release factor RF1, indicating that the reaction mechanism of the two structurally very similar proteins must be different. In addition, the position 1916 of 23S rRNA was found to be essential in determining the specificity of the pseudouridine synthase RluD for helix 69.
The aim of the present dissertation was to analyze i) the role of helix 69 of the ribosomal 23S rRNA in the the factor-catalyzed steps of protein synthesis and ii) the role of pseudouridines in helix 69 for translation termination. The factors determining the substrate specificity of the pseudouridine synthase RluD were also investigated. The involvement of helix 69 in ribosomal functioning has been suggested by x-ray and cryo-EM analyses of various ribosomal complexes. However, at the beginning of the present work the majority of the structure-based suggestions about the regulatory role of helix 69 had not been biochemically tested. In our studies, selected residues of helix 69 were replaced by site-directed mutagenesis, the variant ribosomes were expressed in E.coli in the presence of WT ribosomes and affinity-purified and tested for their activity in different in vitro assays. Our results indicate that helix 69 participates in the regulation of the activity of the initiation factors during the 70S initiation complex formation and is important for the transition of the ribosome from initiation to processive elongation. Depletion of pseudouridines from helix 69 impaired peptide release by release factor RF2 but not by release factor RF1, indicating that the reaction mechanism of the two structurally very similar proteins must be different. In addition, the position 1916 of 23S rRNA was found to be essential in determining the specificity of the pseudouridine synthase RluD for helix 69.
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Väitekirja elektrooniline versioon ei sisalda publikatsioone.
Keywords
dissertatsioonid, bioloogia, biokeemia, valgusüntees, mutagenees