Bacterial Functions Involved in Cell Growth Control

参与细胞生长控制的细菌功能

• 批准号: 6762023
• 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/6762023
• 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; phosphorylation; protein biosynthesis; protein degradation; stress proteins; transcription factor

We have focused our studies on two types of post-transcriptional regulation of gene expression, regulation of mRNA degradation and translation by small regulatory RNAs and regulation of protein stability by energy-dependent proteases. RpoS, a central stress response regulator in Escherichia coli, is subject to both of these levels of control. Degradation of RpoS requires ClpXP protease, and RssB, a protein that presents RpoS to the protease. Degradation is signaled by phosphorylation of RssB; we are investigating the mechanism of phosphorylation and dephosphorylation in vivo and in vitro. Deletion analysis of RssB indicates that sequences at the C-terminus are critical for proper release of the substrate to the protease; N-terminal sequences are required for interaction with the protease. RpoS translation is positively regulated by at least two small RNAs. The message upstream of the RpoS translation start folds into a hairpin that occludes ribosome binding and therefore translation. The small regulatory RNAs, DsrA and RprA, compete for the inhibitory stem of the hairpin, disrupting 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; while many elements in this region contribute to the temperature regulation, the RNA polymerase interaction site at -10 is most critical. Our results suggest that changes in promoter structure may be mediating temperature regulation. RprA, identified as a multicopy suppressor of dsrAmutants, 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. It seems possible that this small RNA is particularly important during biofilm formation. A collaboration with Dr. Gisela Storz used our knowledge of the small RNAs described above to develop a strategy for finding novel small RNAs in E. coli. This search resulted in the identification of 17 new small RNAs and six new, small ORFs. Different small RNAs are expressed under different growth conditions, and about half of them bind well to an RNA chaperone, Hfq, that is also necessary for DsrA and RprA action. One of these novel small RNAs, RyhB, has been investigated in some detail. We find that it is repressed by the Fur, iron-dependent repressor, and is therefore made in high quantities when intracellular iron is limiting. When it is made, it targets multiple mRNAs for degradation. The target mRNAs encode either iron storage proteins (ferritins) or iron-containing but non-essential metabolic proteins. Therefore, this small RNA, which is also found in Vibrio, Salmonella, Klebsiella, and Yersinia, reprograms iron use in the cells and may be an important component of virulence for some pathogens. The other novel small RNAs are likely to represent equally interesting new regulatory pathways. In addition, the information gained from our genome-wide search for small RNAs in E. coli is forming the basis for extending the search for small RNAs to other bacterial species.

我们的研究集中在两种类型的基因表达的转录后调节，小的调节RNA的mRNA降解和翻译的调节和能量依赖性蛋白酶的蛋白质稳定性的调节。RpoS是大肠杆菌中的一种中枢应激反应调节因子，受这两种水平的控制。RpoS的降解需要ClpXP蛋白酶和RssB，RssB是将RpoS呈递给蛋白酶的蛋白质。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。RssB的缺失分析表明，C-末端的序列对于底物向蛋白酶的适当释放至关重要; N-末端序列是与蛋白酶相互作用所需的。RpoS翻译受至少两个小RNA的正调控。RpoS翻译起始点上游的信息折叠成发夹，其阻断核糖体结合并因此阻断翻译。小的调控RNA，DsrA和RprA，竞争发夹的抑制茎，破坏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，而在这个区域中的许多元件有助于温度调节，RNA聚合酶的相互作用位点在-10是最关键的。我们的研究结果表明，启动子结构的变化可能是介导的温度调节。RprA被鉴定为dsrA突变体的多拷贝抑制子，由两个组分RcsC和RcsB调节子调节。这些调节因子也可用于增加荚膜多糖的合成，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。这种小RNA在生物膜形成过程中似乎特别重要。 与Gisela Storz博士合作，利用我们对上述小RNA的了解，开发了一种在大肠杆菌中发现新型小RNA的策略。杆菌这一搜索导致了17个新的小RNA和6个新的小ORF的鉴定。不同的小RNA在不同的生长条件下表达，其中约一半与RNA伴侣Hfq结合良好，这也是DsrA和RprA作用所必需的。这些新的小RNA之一，RyhB，已经进行了一些详细的研究。我们发现，它是抑制的毛皮，铁依赖性阻遏物，因此，在大量的细胞内铁是有限的。当它被制造时，它靶向多个mRNA进行降解。靶mRNA编码铁储存蛋白（铁蛋白）或含铁但非必需的代谢蛋白。因此，这种小RNA也存在于弧菌、沙门氏菌、克雷伯氏菌和耶尔森氏菌中，重新编程细胞中的铁使用，可能是某些病原体毒力的重要组成部分。其他新的小RNA可能代表同样有趣的新调控途径。此外，我们在E.大肠杆菌的研究为将小RNA的研究扩展到其他细菌物种奠定了基础。