Grants and Contributions:

Grant or Award spanning more than one fiscal year. (2017-2018 to 2022-2023)

The incorporation of the 21st amino acid selenocysteine (Sec) into proteins in mammalian cells is guided by the selenosome. The function of this protein complex requires a multitude of protein-protein and protein-RNA interactions leading to the incorporation of selenocysteine at UGA codons, guided by stem-loop structures in the 3’-non translated regions of the selenoprotein-encoding genes. The long-term objective of our research program is to understand the mechanism of Sec incorporation into proteins. This process is of vital importance for mammalian cells and for the metabolism of many bacteria, e.g. for bacterial fermentations. In our preliminary work we have studied the interactions between all the known components of the selenosome using an in vivo assay that is based on bioluminescence resonance energy transfer (BRET). We have also used biochemical approaches to validate and to further characterize these interactions using biochemical and genetic approaches leading to the discovery of novel interactions among components of the Sec biosynthesis machinery. These results led to the guiding hypothesis of our work for the next funding period: A sequence of protein-protein interactions between SPS1, SPS2, SecS and SECp43 guides the incorporation of selenium into its specific tRNA. These reactions are followed by transfer of Sec to the elongation factor and incorporation into the growing polypeptide chain. We here propose to study these early steps of the Sec incorporation pathway at high resolution using cutting edge approaches of biochemistry, cell biology, molecular biology and structural biology.

Specific aim 1. Characterizing the interaction between SPS1, SPS2, SecS and SECp43 using a combination of phage display and biochemical approaches.

Specific aim 2. Atomic resolution-level analysis of the SPS1-SPS2-SecS-SECp43 interaction using X-ray crystallography and single-particle cryo-electron microscopy.

Specific aim 3. In vivo analysis of the dynamic interactions between SPS1, SPS2, SecS, SECp43 and other selenosome components inside mammalian cells.

Our long-term program will reveal the molecular basis for the precise incorporation of Sec into proteins and we will obtain atomic resolution structures of the selenosome. Also, we will visualize Sec incorporation in living cells using highest resolution imaging techniques. More detailed understanding of the molecular basis of this process will enable us to modify it in future to incorporate unnatural amino acids intro proteins, which has very interesting applications in protein engineering and synthetic biology.