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Molecular mechanism of selenocysteine incorporation in bacterial translation

dc.contributor.advisorRodnina, Marina Prof. Dr.de
dc.contributor.authorKotini, Suresh Babude
dc.date.accessioned2012-06-27T18:35:38Zde
dc.date.accessioned2013-01-18T14:23:30Zde
dc.date.available2013-01-30T23:51:09Zde
dc.date.issued2012-06-27de
dc.identifier.urihttp://hdl.handle.net/11858/00-1735-0000-000D-F0BB-9de
dc.identifier.urihttp://dx.doi.org/10.53846/goediss-3190
dc.format.mimetypeapplication/pdfde
dc.language.isoengde
dc.rights.urihttp://creativecommons.org/licenses/by-nc-nd/3.0/de
dc.titleMolecular mechanism of selenocysteine incorporation in bacterial translationde
dc.typedoctoralThesisde
dc.title.translatedEnglishde
dc.contributor.refereeRodnina, Marina Prof. Dr.de
dc.date.examination2011-06-27de
dc.subject.dnb570 Biowissenschaftende
dc.subject.dnbBiologiede
dc.subject.gokWF 200de
dc.description.abstractengSelenocysteine is the 21st amino acid which is incorporated into proteins by recoding a stop codon UGA followed by a selenocysteine insertion sequence (SECIS) of the mRNA. In bacteria, selenocysteine insertion requires specialized machinery which includes selenocysteine-specific tRNASec, translation factor SelB which delivers Sec-tRNASec to the ribosome, as well as proteins SelA, SelD, and seryl-tRNA synthetase which are required to produce Sec-tRNASec. The aim of this work is to develop experimental assays to study Sec incorporation into proteins in vivo and in vitro. As an in vivo assay, we designed the dual luciferase reporter assay and validated its performance using Western blots and luciferase reactions. The efficiency of Sec incorporation was 35% independent of growth conditions. Rapidly growing cells required additional selenium source for efficient Sec insertion. This level of UGA recoding could be reproduced in the fully reconstituted in vitro translation system upon synthesis of a fragment of a natural selenoprotein FdfH. The recruitment of SelB to the SECIS-element prior to translation prevented inhibition of Sec insertion by RF2, a termination factor which usually recognizes the UGA codon and competes with Sec-tRNASec for binding to the ribosome. These results shed light on the importance of the SECIS and on the mechanism by which a stop codon is redirected for efficient readthrough by a specific tRNA.de
dc.contributor.coRefereeStark, Holger Prof. Dr.de
dc.contributor.thirdRefereeFicner, Ralf Prof. Dr.de
dc.subject.topicGöttingen Graduate School for Neurosciences and Molecular Biosciences (GGNB)de
dc.subject.engselenocysteinede
dc.subject.engSECIS-elementde
dc.subject.engin vivode
dc.subject.engin vitrode
dc.subject.engtranslationde
dc.subject.engribosomede
dc.subject.engselBde
dc.subject.bk42.00de
dc.identifier.urnurn:nbn:de:gbv:7-webdoc-3587-0de
dc.identifier.purlwebdoc-3587de
dc.affiliation.instituteGöttinger Graduiertenschule für Neurowissenschaften und Molekulare Biowissenschaften (GGNB)de
dc.identifier.ppn730380807de


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