Bacterial Functions Involved in Cell Growth Control

Gottesman项目名称概要我们以大肠杆菌为模型系统，继续研究能量依赖性蛋白质降解在基因表达调控中的作用。 对ClpAP蛋白酶的ClpA ATP酶的诱变研究已经鉴定了ATP酶结构域内的保守残基中的突变，这些突变产生显性负表型和干扰ClpXP底物降解的其他突变。 这些突变的生化性质的分析应该提供进一步的洞察底物是如何识别和处理这种蛋白酶。 RssB是一种调节固定相σ因子RpoS降解的蛋白质，已发现其将RpoS呈递给ClpXP蛋白酶。降解是通过RssB的磷酸化发出信号的;我们正在研究体内和体外磷酸化和去磷酸化的机制。 除了RpoS降解的调节外，RpoS翻译还受DsrA（一种稳定的85个核苷酸的小RNA）的调节。 RNA 5'端的茎环是RpoS调控所必需的，并通过与RpoS前导序列配对起作用。这破坏了RpoS前导序列的二级结构，允许翻译。 dsrA的启动子受温度调节（在低温下开启，在高温下关闭）。 我们发现，温度调节存在于36个碱基对的最小启动子区域，并且可能在+1到10个碱基对的区域内。 15，表明启动子结构可能介导温度调节。 一种新的RNA RprA被鉴定为dsrA突变体的抑制因子。 它还可以刺激RpoS翻译，尽管其作用机制似乎与DsrA不同。 DsrA和RprA均参与RpoS的渗透压休克诱导。 RprA的合成受两种组分RcsC和RcsB调节剂的调节。这些调节因子也可以促进荚膜多糖的合成，并上调细胞分裂蛋白;它们被细胞表面应激激活。 当它们被激活时，RpoS合成以RprA依赖性方式增加。 除了DsrA之外，RpoS的第二个小RNA调节子的证明支持了多个小RNA在许多情况下可能介导翻译调节的想法。 与Gisela Storz博士的合作已经开始从E.大肠杆菌，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.