BACTERIAL FUNCTIONS INVOLVED IN CELL GROWTH CONTROL

Gottesman项目标题摘要我们继续研究能量依赖性蛋白质降解在调节基因表达中的作用，使用大肠杆菌作为模型系统。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。RssB的磷酸化是降解的信号;我们正在研究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，温度调节驻留在一个最小的启动子区域的36个碱基对，这表明启动子结构可能介导的温度调节。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。渗透压休克似乎调节它们的作用而不是它们的合成。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。 在Lon蛋白酶的诱变研究中，我们发现C-末端结构域中的缺失和点突变去除或去除蛋白水解活性位点，当过量产生时，仍然能够部分地补充Lon突变体。这似乎是因为底物结合结构域保留在缺失蛋白的N-末端，底物的结合足以干扰底物功能，模拟降解效应。然而，与缺失的蛋白质的结合保护底物免受二级蛋白酶的影响。这将允许由Lon蛋白酶的底物识别和结合的分析独立于蛋白质降解进行研究。通过部分蛋白水解和通过使用蛋白酶敏感性区域作为单独表达的Lon结构域的边界来进行Lon结构域的进一步分析。通过分析这些片段的体内和体外性质，我们开始了解这种能量依赖性蛋白酶的组织。类似的方法被用于分析ClpAP蛋白酶的ClpA ATP酶。 - 大肠杆菌，基因调控，热休克蛋白，原核生物，蛋白酶，转录控制，调节RNA，-既不是人类受试者也不是人类组织

Gottesman Project Title SummaryWe have continued studies on the role that energy-dependent protein degradation plays in regulating gene expression, using Escherichia colias a model system. 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 signalled by phosphorylation of RssB; we are investigating the mechanism of phosphorylation and dephosphorylation. 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 dsrAis 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, suggesting that promoter structures may be mediating temperature regulation. A novel new RNA, RprA, has been identified as a suppressor of dsrAmutants. 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. Osmotic shock appears to modulate their action rather than their synthesis. 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. In mutagenesis studies of the Lon protease, we find that deletions and point mutations in the C- terminal domain that remove or inactivate the proteolytic active site are still able to partially complement lon mutants when overproduced. This appears to be because the substrate binding domain is retained in the N-terminus of the deleted proteins, and binding of substrate is sufficient to interfere with substrate function, mimicking the effect of degradation. However, binding to the deleted proteins protects substrates from secondary proteases. This will allow the analysis of substrate recognition and binding by the Lon protease to be studied independently of protein degradation. Further analysis of Lon domains was carried out by partial proteolysis and by using regions of protease sensitivity as the boundaries for separately expressed Lon domains. By analysis of the in vivo and in vitro properties of these fragments, we are beginning to understand the organization of this energy-dependent protease. Similar approaches are being used to analyze the ClpA ATPase of the ClpAP protease. - Escherichia coli, Gene regulation, heat shock proteins, prokaryotes, proteases, transcriptional control, regulatory RNA, - Neither Human Subjects nor Human Tissues