Structural and functional investigation of tRNA guanine transglycosylase

by Katharina Sievers
Cumulative thesis
Date of Examination:2023-06-09
Date of issue:2023-11-17
Advisor:Prof. Dr. Ralf Ficner
Referee:Prof. Dr. Ralf Ficner
Referee:Prof. Dr. Markus Bohnsack
Persistent Address: http://dx.doi.org/10.53846/goediss-10207

 

 

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Abstract

English

In this thesis, two novel structures were presented, jointly illuminating dimerization and tRNA binding by human TGT. A first structure, obtained by X-ray crystallography, allowed to give a thorough account of the atomic details of the heterodimeric interface as well as active site recognition of the substrate RNA. The second structure, obtained by single particle cryo-EM, is the first to show complete tRNA binding by a TGT enzyme of the bacterial/eukaryotic type, revealing that both TGT subunits are intimately involved in tRNA binding. Together, these two structures close several knowledge gaps that remained in the understanding of eukaryotic TGT and the broader TGT family. Furthermore, insights on TGT flexibility yielded by cryo-EM dataset were expanded on by solution small-angle X-ray scattering, allowing to move beyond the previously rigid, crystal-based view on TGT. The structural data of this thesis was further accompanied by biochemical characterization, focusing on tRNA binding and the association between tRNA body and non-catalytic subunit. This integrative approach yielded a new understanding of eukTGT function which, by careful comparison with bacterial and archaeal TGT, was finally placed in an evolutionary context. In consequence, the structural biology of eukaryotic TGT and its tRNA complex is now well characterized. In contrast, TGT subcellular location, regulation and potential transport are all largely unknown. It remains to be hoped that complimentary approaches will shed light on these issues in the future and thus grant a full understanding of eukaryotic TGT in its cellular context.
Keywords: tRNA; RNA-binding; crystallography; RNA-modification
 
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