Bacterial Functions Involved in Cell Growth Control

Gottesman项目标题摘要我们使用大肠杆菌作为模型系统，继续研究依赖能量的蛋白质降解在调节基因表达中所起的作用。对ClpAP蛋白酶ClpA ATPase的突变研究发现，ATPase结构域中保守残基发生突变，导致显性负表型和其他干扰ClpXP底物降解的突变。对这些突变的生化性质的分析应该提供对底物如何被这种蛋白酶识别和处理的进一步洞察。RSSB是一种调节固定相sigma因子rpos降解的蛋白质，已被发现向ClpXP蛋白酶递送rpos。降解是通过RSSB的磷酸化来表示的；我们正在研究体内和体外的磷酸化和去磷酸化的机制。除了调节rpos的降解外，rpos的翻译还受DSRA的调控，DSRA是一种稳定的85个核苷酸的小RNA。RNA 5‘端的茎环是调节RPOS所必需的，它通过与RPOS先导配对而发挥作用。这打乱了RPOS领导者的二级结构，允许进行翻译。DSRA启动子受温度调节(低温开启，高温关闭)。我们发现温度调节位于36个碱基对的最小启动子区域，可能在+1到15之间，这表明启动子结构可能介导温度调节。一种新的RNA，RPRA，已被鉴定为DSRA突变体的抑制因子。它还起到刺激RPOS翻译的作用，尽管其作用机制似乎与DSRA不同。DSRA和RPRA均参与rpos的渗透休克诱导。RPRA的合成受RCSC和RCSB两种调节剂的调节。这些调节剂还可以促进被膜多糖的合成，并上调细胞分裂蛋白；它们被细胞表面的压力激活。当它们被激活时，RPOS合成以RPRA依赖的方式增加。除DSRA外，RPOS的第二个小RNA调节子的证明支持这样的观点，即在许多情况下，多个小RNA可能介导翻译调节。一项与Gisela Storz博士的合作已经开始从这些和其他已知的大肠杆菌小RNA中推断，以确定检测其他新的小RNA的条件。

GottesmanProject Title Summary We have continued studies on the role that energy-dependent protein degradation plays in regulating gene expression, using Escherichia coli as a model system. Mutagenesis studies on the ClpA ATPase of the ClpAP protease have identified mutations in conserved residues within the ATPase domains that give dominant negative phenotypes and others that perturb degradation of a ClpXP substrate. Analysis of the biochemical nature of these mutations should provide further insight into how substrates are recognized and processed by this protease. RssB, a protein that regulates the degradation of the stationary phase sigma factor RpoS has been found to present RpoS to the ClpXP protease. Degradation is signaled by phosphorylation of RssB; we are investigating the mechanism of phosphorylation and dephosphorylation in vivo and in vitro. In addition to regulation of RpoS degradation, RpoS translation is regulated by DsrA, a small stable 85 nucleotide RNA. A stem-loop at the 5' end of the RNA is necessary for regulation of RpoS, and acts by pairing with the RpoS leader. This disrupts the secondary structure of the RpoS leader, allowing translation. The promoter of dsrA is regulated by temperature (on at low temperatures, off at high temperatures). We find that temperature regulation resides in a minimal promoter region of 36 base pairs, and probably within the region from +1 to 15, suggesting that promoter structures may be mediating temperature regulation. A novel new RNA, RprA, has been identified as a suppressor of dsrA mutants. It also acts to stimulate RpoS translation, although the mechanism of action appears to differ from that for DsrA. Both DsrA and RprA participate in the osmotic shock induction of RpoS. RprA synthesis is regulated by the two component RcsC and RcsB regulators. These regulators also act to turn up capsular polysaccharide synthesis and to up regulate a cell division protein; they are activated by cell surface stress. When they are activated, RpoS synthesis increases in an RprA-dependent fashion. The demonstration of a second small RNA regulator of RpoS, in addition to DsrA, supports the idea that multiple small RNAs may mediate translational regulation in many cases. A collaboration with Dr. Gisela Storz has been started to extrapolate from these and other known small RNAs in E. coli to identify conditions for detecting other novel small RNAs.