Functional analysis of the tRNA binding protein Kti12

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By operating in tRNA modification, the Elongator complex (Elp1-Elp6) assures accurate mRNA decoding during translation. For proper expression of genetic information, one may therefore expect Elongator to be permanently active. Evidence from several research teams including our own, however, shows that tRNA modifications do change and that there are accessory Elongator partner proteins (Kti11-Kti14). This suggests the activity of the modifier complex is regulated rather than constitutive. In line with this notion, Elongator’s largest subunit (Elp1) undergoes dynamic de/phosphorylation by a kinase (Hrr25/Kti14) and a phosphatase (Sit4). In addition, we have shown through DFG support that Elongator partner protein Kti12 apparently is required to promote recruitment of kinase Hrr25 for activating phosphorylation of the Elongator complex.In recent work funded by the DFG, we discovered by crystalization as well as in-depth genetic and biochemical analyses that Kti12 is a structural mimic of a tRNA-Kinase (PSTK), hydrolyses NTPs with preference for ATP and binds tRNAs with an affinity lower than Elongator. How precisely Kti12 interacts with Elongator or Hrr25 and whether its capacity to bind tRNA or hydrolyze ATP promotes the tRNA modification activity of the Elongator complex are key aspects we aim to address in this project proposal in the following ways:- analysis of the role tRNA binding by Kti12 plays for tRNA modification by Elongator using structure-guided KTI12 mutagenesis for generation of kti12 mutants with tRNA binding defects- analysis of the role ATPase activity of Kti12 plays for Elongator function using wild-type Kti12 and ATPase mutants in Elongator and tRNA interaction assays with nucleotides or non-hydrolyzable analoga- analysis of the Elongator activator function of Kti12 as part of a protein network using Elongator interaction, phosphorylation and tRNA modification assays with wild-type Kti12 or tRNA binding and ATPase mutants- identification of Elongator unrelated Kti12 interactors by MALDI-TOF-MS/MS after purification of Elongator-free Kti12 pools from wild-type cells and mutants with tRNA binding or ATPase defectsIn sum, our proposed project will use in vivo & in vitro methods aimed at identifying Kti12 structure-function requirements to enhance our understanding about the regulation of the tRNA modifier complex Elongator. With a growing body of evidence showing that Elongator linked tRNA modification defects associate with mistranslation, proteotoxicity and formation of neuropathies in humans, our project has biomedical relevance. Thus, a better knowledge about the roles played by inappropriate tRNA modifications under conditions of Elongator dysfunction may provide insights into disease syndrome mechanisms and inform therapeutic intervention schemes.