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

在第一个授予期间，我们已经证明了通过终止-重新起始(Tere)的翻译偶联(Tere)对于模式考古菌Haloferax Volcanii和模式细菌E.Coli中重叠的基因对是高效的。Shine Dalgarno基序对Tere效率的重要性具有高度的变异性和基因特异性。TERE的效率关键取决于基因重叠或小于30个核苷酸的极短基因间隔区。在第二个授权期，我们将对导致有效Tere的序列和结构基序进行深入分析，包括在H.Volcanii和E.Coli的基因重叠附近进行饱和突变。由于我们已经发现Tere的一个生物学功能可能是有效地形成异构体蛋白质复合体，因此将使用定量Western印迹分析来确定几个基因对火山杆菌和大肠杆菌的绝对Tere效率。体外翻译系统将被用来解开通过Tere的翻译偶联是依赖于30S亚基还是依赖于未解离的70S核糖体。此外，双顺反子转录本上的基因对的表达将与同一基因在两个单顺反子转录本上的表达进行比较，以得出在火山杆菌中是否存在异构体复合体的共翻译形成的指示。综上所述，该项目将对古生物和细菌中通过终止-重新启动的翻译偶联的机制和生物学功能产生前所未有的见解。

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.