Characterization of macromolecular interactions of proteins involved in the selenocysteine synthesis from Escherichia coli

Abstract

The investigation of the genetic code translation to proteins calls the interest for its central role in the cellular metabolism. In particular, the investigation of new amino acid synthesis, such as selenocystreine and pyrolysin that result in the expansion of the genetic code from the traditional 20 amino acid residues to a total of 22 residues.The selenocysteine (Sec, U) represents the major biological form of selenium and its incorporation is a co-translational process in response to a UGA in-frame stop codon and requires a complex enzymatic machinery to be accomplished.The complete set f genes involved in the selenocysteine incorporation in prokaryotes and eukaryotes is known, however, the molecular interactions between the different proteins has not been characterized.This project has the purpose of characterizing at the molecular and structural level the interactions between the Selenocysteine Synthetase (SELA) and the selenocysteine-specific Translation Elongation Factor with the ribosome in Escherichia coli.To achieve these purpose, fluorescence anisotropy and microcalorimetry techniques will be used for the determination of the interaction constants of these protein complexes. To obtain structural models of the protein complex Negative Stain Electron Microscopy, High Resolution Cryo-Electron Microscopy and Hydrogen/Deuterium Exchange Mass Spectroscopy (HDEX) will be employed. The proposed investigation will help to elucidate the mechanism of selencysteine incorporation in bacteria as well as in other domains of life. (AU)