Non-conventional translation initiation in bacteria

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The conventional pathway of translation initiation in prokaryotes relies on the interaction of a Shine Dalgarno (SD) motif with the 3’-end of the 16S rRNA. Recently we could show that translation initiation in haloarchaea is predominated by two non-conventional mechanisms (leaderless transcripts and SD-less transcripts). We have now extended the characterization of translation initiation to Escherichia coli. We could show that SD-less transcripts exist also in E. coli, indicating that non-conventional mechanisms for translation initiation are also used by bacteria. We will use translatome analyses for a global characterization of transcripts with non-average translational efficiencies under stress conditions or kasugamycin treatment. A dual reporter gene system will be used to quantify relative translational efficiencies of the conventional and non-conventional mechanisms under various conditions, and the role of cis-acting elements will be analyzed by mutagenesis. Initiation complexes will be isolated and by using ultrasoft mass spectrometry and proteomics it will be revealed whether specialized complexes exist under different conditions or on non-conventional transcripts (similar to the recently identified specialized 61S ribosomes).