Molecular Mechanisms of Selenocysteine incorporation in Bacterial Bakterial Translation

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Selenocysteine is incorporated into protein on the ribosome when UGA codons, which usually lead to termination, are decoded by Sec-tRNASec, provided a selenocysteine incorporation sequence (SECIS) is present in the mRNA. The project aims at understanding molecular mechanisms of selenocysteine incorporation in the E. coli system. The assembly of SECIS-containing mRNA and the ternary complex SelB·GTP·Sec-tRNASec with the ribosome will be studied kinetically, monitoring fluorescent dyes at various positions of the complex components. The mechanism of GTPase activation of SelB in the ternary complex and its regulation by the mRNA recognition elements, UGA codon and SECIS, will be investigated. Timing and mechanism of SelB dissociation from the ribosome·SECIS-mRNA complex will be studied by fluorescence resonance energy transfer (FRET) between fluorescent groups in SelB and SECIS. Reasons for the low efficiency of in vivo selenocysteine incorporation will be investigated. The experiments will allow us to identify reaction intermediates and determine the kinetics of the respective transitions. In collaboration with H. Stark and M. Wahl, MPI Göttingen, the structure of functional ribosome-SelB complexes and of the SelA·Sec-tRNASec complex will be studied by cryoelectron microscopy and crystallography.