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dc.contributor.advisor Rodnina, Marina Prof. Dr. de
dc.contributor.author Kotini, Suresh Babu de
dc.date.accessioned 2012-06-27T18:35:38Z de
dc.date.accessioned 2013-01-18T14:23:30Z de
dc.date.available 2013-01-30T23:51:09Z de
dc.date.issued 2012-06-27 de
dc.identifier.uri http://hdl.handle.net/11858/00-1735-0000-000D-F0BB-9 de
dc.format.mimetype application/pdf de
dc.language.iso eng de
dc.rights.uri http://creativecommons.org/licenses/by-nc-nd/3.0/ de
dc.title Molecular mechanism of selenocysteine incorporation in bacterial translation de
dc.type doctoralThesis de
dc.title.translated English de
dc.contributor.referee Rodnina, Marina Prof. Dr. de
dc.date.examination 2011-06-27 de
dc.subject.dnb 570 Biowissenschaften de
dc.subject.dnb Biologie de
dc.subject.gok WF 200 de
dc.description.abstracteng Selenocysteine 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.coReferee Stark, Holger Prof. Dr. de
dc.contributor.thirdReferee Ficner, Ralf Prof. Dr. de
dc.subject.topic Göttingen Graduate School for Neurosciences and Molecular Biosciences (GGNB) de
dc.subject.eng selenocysteine de
dc.subject.eng SECIS-element de
dc.subject.eng in vivo de
dc.subject.eng in vitro de
dc.subject.eng translation de
dc.subject.eng ribosome de
dc.subject.eng selB de
dc.subject.bk 42.00 de
dc.identifier.urn urn:nbn:de:gbv:7-webdoc-3587-0 de
dc.identifier.purl webdoc-3587 de
dc.affiliation.institute Göttinger Graduiertenschule für Neurowissenschaften und Molekulare Biowissenschaften (GGNB) de
dc.identifier.ppn 730380807 de

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