Microbiology

Phosphoregulation during cell growth in yeast (Saccharomyces cerevisiae)

On the modulation of Elongator activity through dynamic phosphorylation

Background and previous work

The Elongator complex is responsible for modification of ‘wobble‘ uridine bases (U34) in tRNA anticodons, thereby mediating accurate translation and protein synthesis in yeast (Saccharomyces cerevisiae). Studies have shown that the activity of Elongator is regulated by de-/phosphorylation and a protein network composed of a casein kinase (Hrr25/Kti14), a protein phosphatase (Sit4) and an Elongator partner protein (Kti12). We have identified two phosphorylation sites on Elongator’s largest subunit (Elp1) that are directly phosphorylated by Hrr25. Interestingly, mutations that disrupt the kinase and phosphatase activities of Hrr25 and Sit4, respectively, not only cause opposite Elp1 phosphorylation states but also abolish Elongator-dependent U34 modifications. This suggests that Elongator might require sequential Elp1 phosphorylation cycles for its ability to modify tRNAs. In addition, there is a dynamic component to the phospho-system involving Kti12, a factor supporting Elongator phosphorylation. Moreover, the phosphorylation sites are adjacent to a basic region in Elp1 crucial for tRNA binding. Thus, phosphorylation may have an impact on the ability of Elongator to interact with regulatory proteins and to bind/modify tRNAs.

Specific Aims

Regarding the phosphoregulation of Elongator, we want to focus on individual players of the phospho-system (see above) and characterize their contribution to proper Elongator function and tRNA modification activity.

Working programme

-          Conservation of Hrr25 function in eukaryotes (collaboration with P4)

-          Phosphoregulation of Hrr25 kinase activity (collaboration with P1, P2)

-          Role of Kti12 in kinase/phosphatase dynamics