Structural and functional analysis of the MnmE/ GidA protein complex studied by EPR spectroscopy

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dc.contributor.advisorProf. Dr. Heinz-Jürgen Steinhoff
dc.creatorBöhme, Sabine
dc.description.abstractTransfer RNA (tRNA) molecules play a significant role in the translation of the genetic code into protein sequences. The interaction of the messenger RNA (mRNA) codons with the anticodon of the tRNA at the ribosomal subunit results in the discrimination of cognate versus near-cognate and non-cognate codons. In order to do so post-transcriptional modifications at tRNAs have to occur. Among the ~80 identified modifications, the largest diversity can be found in the anticodon wobble position 34 or immediately 3’ adjacent to the anticodon triplet at position 37. The role of these modifications relies in an efficient protein synthesis, the improvement of reading frame maintenance, and codon recognition on the ribosome. Nevertheless, a wide spectrum of human diseases is caused by a growing number of mutations in mitochondrial tRNA genes, whose phenotypic peculiarities are characterized by a broad spectrum of clinical manifestations, including blindness, deafness, dementia, movement disorders, weakness, cardiac failure, diabetes, renal dysfunction, and liver diseases. To analyze the corresponding mechanism of such diseases, model organisms, e.g. E. coli and S. cerrevisiae are frequently used because of the high degree of evolutionary conservation of the modification pathway. In E. coli, the evolutionary conserved protein MnmE together with its interaction partner GidA are involved in the modification of U34 in the wobble position, which has a pivotal role in the structure and function of tRNAs, including structural stabilization, aminoacylation, and codon recognition at the decoding site of small rRNAs. In this work, the G protein MnmE will be investigated by Electron Paramagnetic Resonance (EPR) Spectroscopy in combination with site-directed spin labeling, together with its interaction partner GidA, during different steps of the G domain cycle, to achieve detailed structural and functional insights of those enzymes and to understand their respective biological functions. Thereby, it could be proven directly for the first time that the G domains contact each other in the presence of the triphosphate (GppNHP) or transition state analogue (GDP·AlFx), while they exhibit an open conformation in the Apo- and GDP-bound state. Moreover, it was possible to demonstrate that only the presence of GDP, AlFx and K+ is capable of stabilizing the closed state, and that this effect is specific, since the effect is absent with Na+ and Cs+ and is smaller with similar size cations such as Rb+ and NH4+. This dependency, however, is partly abolished in the presence of GidA, where the results indicate that interaction with this protein on a site remote from the G domains stabilizes the activated GTPase competent MnmE dimer. Moreover, binding of GidA induces conformational changes in MnmE which in turn mediate dimerization of the G domains even in the absence of potassium ions, indicating that GidA binding significantly alters the relative localization of the G domains and thus acts as a co-stimulator of the GTPase reaction.eng
dc.rightsNamensnennung-NichtKommerziell-KeineBearbeitung 3.0 Unported-
dc.subject.ddc570 - Biowissenschaften; Biologie
dc.subject.ddc530 - Physik
dc.titleStructural and functional analysis of the MnmE/ GidA protein complex studied by EPR spectroscopyeng
dc.typeDissertation oder Habilitation [doctoralThesis]-
thesis.typeDissertation [thesis.doctoral]-
dc.contributor.refereeapl. Prof. Dr. Richard Wagner
Appears in Collections:FB05 - E-Dissertationen

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