BACTERIAL FUNCTIONS INVOLVED IN CELL GROWTH CONTROL

参与细胞生长控制的细菌功能

• 批准号: 6289209
• 负责人: SUSAN GOTTESMAN
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6289209
• 关键词: Escherichia coli; bacterial genetics; cell growth regulation; endopeptidases; enzyme activity; enzyme substrate; gene environment interaction; gene expression; genetic regulation; genetic translation; molecular cloning; protein biosynthesis; protein degradation; stress proteins; transcription factor

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

Gottesman 项目标题摘要我们使用大肠杆菌作为模型系统，继续研究能量依赖性蛋白质降解在调节基因表达中的作用。 RssB 是一种调节稳定期 sigma 因子 RpoS 降解的蛋白质，已被发现将 RpoS 呈递给 ClpXP 蛋白酶。降解由 RssB 磷酸化发出；我们正在研究磷酸化和去磷酸化的机制。除了调节 RpoS 降解外，RpoS 翻译还受 DsrA（一种稳定的 85 个核苷酸的小 RNA）调节。 RNA 5 端的茎环对于 RpoS 的调节是必需的，并通过与 RpoS 前导序列配对发挥作用。这会破坏 RpoS 领导者的二级结构，从而允许翻译。 dsrA 的启动子受温度调节（低温时开启，高温时关闭）。我们发现温度调节存在于 36 个碱基对的最小启动子区域，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 dsrAmutants 的抑制因子。它还能刺激 RpoS 翻译，但作用机制似乎与 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 调节因子的论证支持了多个小 RNA 在许多情况下可能介导翻译调节的观点。 在 Lon 蛋白酶的诱变研究中，我们发现 C 端结构域中删除或使蛋白水解活性位点失活的缺失和点突变在过量产生时仍然能够部分补充 lon 突变体。这似乎是因为底物结合结构域保留在删除的蛋白质的 N 末端，并且底物的结合足以干扰底物功能，模拟降解的效果。然而，与删除的蛋白质的结合可以保护底物免受次级蛋白酶的影响。这将使 Lon 蛋白酶的底物识别和结合分析能够独立于蛋白质降解进行研究。通过部分蛋白水解并使用蛋白酶敏感性区域作为单独表达的 Lon 结构域的边界，对 Lon 结构域进行了进一步分析。通过分析这些片段的体内和体外特性，我们开始了解这种能量依赖性蛋白酶的组织。类似的方法也被用于分析 ClpAP 蛋白酶的 ClpA ATPase。 - 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