| dc.description.abstracteng | Posttranslational and posttranscriptional modifications have long been known to modulate and extend the properties of proteins and nucleic acids. A vast variety of non-canonical bases have been found on RNA molecules, with a huge number of them occurring on transfer RNAs (tRNAs).Here they modulate the molecule’s stability, chemical properties or even its shape, which are crucial elements for the correct function of tRNAs. In contrast to modifications that have been described on proteins like histones, where a close interplay of modifications has been elucidated, least is known about how RNA modifications affect each other and whether they may be linked. Considering an average of 14 modifications occurring on an tRNA molecule simultaneously, the study of an eventual link between these modifications becomes imminent. In contrast to the numerous studies focusing on a single modification, recent publications have presented a link between the 7deaza-guanosine derivative queuosine (Q), which occurs on the tRNAAsp wobble base 34 (Q34), and Dnmt2 mediated methylation of the C38 base (m5C38) in a downstream manner. While investigation of this linkage identified C38 methylation to strongly depend on presence of queuosine in the tRNA target, least in known about the underlying molecular mechanism of this interplay.
Prior to this thesis, the structure of the tRNA guanine transglycosylase (TGT), which establishes Q34 modification by incorporating the modified queuine base into the tRNAs tRNAAsp, tRNAAsn, tRNAHis and tRNATyr, was unknown. Within this work the first structure of the catalytic subunit QTRT1 of the TGT heterodimer is reported. Investigation of this structure of the human QTRT1 reveals a high conservation, suggesting the reaction mechanism to be conserved from bacteria to men. Furthermore, the QTRT1 structure was solved in complex with the queuine base providing first insights into the accommodation of this hypermodified base. Within this thesis, the newly solved QTRT1 crystal structure is further investigated with focus on RNA interaction and phosphorylation, latter of which is implicated in TGT activity.
In a second part, the Dnmt2 methyltransferase is investigated biochemically and structurally with focus on Q34 substrate modification, as the dependence of tRNAAsp C38 methylation in S. pombe has been shown to depend on the presence of TGT reaction product queuosine as part of the substrate in vivo. However, the underlying mechanism how queuosine modulates the activity of the m5C38 depositing enzyme Dnmt2 is not known. As part of this work, queuine modification of tRNAAsp alone is found to be sufficient to trigger Dnmt2 activity in vitro. Furthermore, a model of Dnmt2 tRNA substrate complex was generated by computational docking of the tRNA to the newly solved S. pombe Dnmt2 crystal structure. Combination of biochemical and structural data lead to the conclusion that triggering of Dnmt2 activity by Q34 is mediated by optimal positioning of the relevant reaction components.
Substrate specificity of Dnmt2 is further investigated and set into relation with the S. pombe Dnmt2 structure as well as previously deposited Dnmt2 structures. Furthermore, the docked Dnmt2 tRNA complex is found to be in high agreement with cross link data and identified as the most advanced model of Dnmt2 substrate interaction. Finally, a working model for Dnmt2 mediated methylation is proposed by combining data reported as part of this work with the other reported studies on the Dnmt2 enzyme. | de |