Processing and degradation of regulatory RNAs in Escherichia coli

为了理解RNA的调控，有必要知道这些RNA分子是如何产生和翻转的。虽然一些小RNA代表初级转录物，但其他RNA是由较大的RNA转录物通过加工产生的。一些小RNA通过影响靶RNA的稳定性来发挥其功能。即使小RNA不影响靶RNA的衰变，其自身的周转率也可能强烈影响其调控作用。虽然一些小rna非常稳定（半衰期为30分钟或更长），但其他rna在几分钟内就会被翻转。最初，我们将集中研究三个系统：i) ibpBA mRNA，其中包含两个假设的RNA温标，Franz Narberhaus对其功能进行了分析；ii) rpoS和影响rpoS表达的小RNA，涉及固定相和应激调节（由R. Hengge和J. Vogel研究）；iii) GcvB和Omr RNA（影响外膜组成），它们表现出明显不同的半衰期。rna的衰变将在不同的生长条件下和RNase突变株中进行分析，以便初步了解潜在的机制。这将通过使用体外转录物与靶RNA和/或RNA伴侣Hfq以及分离的RNA酶或降解体复合物结合的体外研究加以补充。对这些研究中产生的衰变机制的假设将通过产生修饰的rna来挑战，其衰变将再次在体内和体外进行分析。

To understand the regulation by RNA it is necessary to know how these RNA molecules are generated and turned-over. While some small RNAs represent primary transcripts, others are generated from larger RNA transcripts by processing. Some small RNAs exert their function by influencing the stability of the target RNA. Even if the small RNA does not influence the decay of target RNAs, its own turn-over rate may strongly influence its regulatory impact. While some small RNAs are very stable (half-life 30 min or more), others are turned-over within few minutes. Initially we will concentrate our studies on three systems: i) the ibpBA mRNA which harbours two putative RNA thermometers that are analyzed in regard to function by Franz Narberhaus, ii) rpoS and the small RNAs influencing rpoS expression involved in stationary phase and stress regulation (studied by R. Hengge and J. Vogel) and iii) the GcvB and Omr RNAs (affect outer membrane composition) which exhibit remarkably different half-lives. The decay of the RNAs will be analyzed under different growth conditions and in RNase mutant strains in order to get first insights into the underlying mechanisms. This will be complemented by in vitro studies using in vitro transcripts in combination with target RNAs and/or the RNA chaperone Hfq and isolated RNases or degradosome complexes. Hypotheses on the decay mechanisms arising from these studies will then be challenged by generating modified RNAs, whose decay will again be analyzed in vivo and in vitro.