Transational coupling via termination-reinitiation at overlapping genes in archaea and bacteria

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In the first granting period, we have shown that translational coupling via termination-reinitiation (TeRe) is highly effective at overlapping gene pairs in the model archaeon Haloferax volcanii and the model bacterium Escherichia coli. The importance of the Shine Dalgarno motif for the efficiency of TeRe is highly variable and gene specific. The efficiency of TeRe critically depends on gene overlaps or extremely short intergenic regions of less than 30 nt. In the second granting period, we will perform in depth analyses of the sequence and structural motifs that lead to efficient TeRe, including saturation mutagenesis near gene overlaps in H. volcanii and E. coli. As we have found indications that one biological function of TeRe might be the efficient formation of heteromeric protein complexes, quantitative Western blot analyses will be used to determine the absolute TeRe efficiencies for several gene pairs of H. volcanii and E. coli. An in vitro translation system will be used to unravel whether translational coupling via TeRe depends on the 30S subunit or on the undissociated 70S ribosome. In addition, expression from gene pairs on bicistronic transcripts will be compared with expression of the same genes on two monocistronic transcripts to yield indications whether co-translational formation of heteromeric complexes occurs in H. volcanii. Taken together, the project will yield unprecedented insight into the mechanism and biological function of translational coupling via termination-reinitiation in archaea and bacteria.