Cells employ ribosomes and transfer RNA (tRNA) molecules to translate sequence information encoded in mRNA and produce correctly-folded proteins, a process aided by the highly conserved Elongator complex whose modifying activity on tRNA is essential for maintaining accurate translational rates, protein folding dynamics, global protein homeostasis and altogether keeping many severe human diseases at bay. Further regulatory mechanisms involve various post-transcriptional RNA modifications, currently encompassed by the term “epitranscriptome”.
We propose to dissect the molecular mechanisms that allow the Elongator complex to conduct specific uridine bases modifications in the wobble base position of tRNAs.
We plan to combine cryo-electron microscopy with detailed mechanistic and functional studies using protein biochemistry, biophysics and in vivo validation.
We will use complementary protein-protein and protein-tRNA interaction assays in vitro to define stable entities suitable for homogenous vitrification, which will validate our structural information to, in turn, produce structure-guided mutations.
We will obtain high-res structures of the fully assembled Elongator complex bound to endogenous target tRNAs decorated with different combinations of other types of tRNA modifications and determine the structures of the thiolation cascade components bound to the Elongator complex which are recruited via one of its regulatory factors.We will test the interaction of the Elongator complex with various kinases, analyze the influence of the resulting phosphorylation events and determine the structure of the identified complexes.
We will test the interaction of the Elongator complex with various kinases, analyze the influence of the resulting phosphorylation events and determine the structure of the identified complexes.
The results of our mechanistic experiments will be confirmed by our long-term collaborators at German and Swiss research institutes who will test functionally relevant mutants in established phenotypical assays in vivo using model organisms like yeast, fish and worms.
Our research will provide deep molecular and functional insights into so far poorly characterized regulatory networks for protein synthesis and proteome homeostasis. Gaining this understanding into the Elongator complex cellular mechanisms is also of prime clinical importance as the occurrence of specific mutations in different Elongator subunits in patients is directly correlated with the onset of neurodegenerative diseases and the appearance of treatment-resistant cancer cells, making them potential diagnostic markers and intervention points for targeted therapies.
Edited by: Dr. A. Flores-Ibarra
