Bacterial Functions Involved in Cell Growth Control

我们主要研究两种类型的基因表达转录后调控，小调控rna对mRNA降解和翻译的调控以及能量依赖性蛋白酶对蛋白质稳定性的调控。RpoS是大肠杆菌中的一种中央应激反应调节因子，受这两种水平的控制。RpoS的降解需要ClpXP蛋白酶和RssB，一种将RpoS呈现给蛋白酶的蛋白质。降解是由RssB的磷酸化信号；我们正在体内和体外研究磷酸化和去磷酸化的机制。RssB的缺失分析表明，c端序列对底物向蛋白酶的适当释放至关重要；与蛋白酶相互作用需要n端序列。

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 activate synthesis of 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, and more recently to find a particular class of small RNAs that bind an RNA chaperone, Hfq. The initial search resulted in the identification of 17 new small RNAs and six new, small ORFs. Different small RNAs are expressed under different growth conditions. Hfq, an RNA chaperone that is necessary for DsrA and RprA action, was found to bind to about 1/3 of the previously known small RNAs in E. coli, and potentially 20 new small RNAs were identified by their binding to Hfq. Hfq stabilizes the small RNAs and helps them pair to mRNA targets. Once paired to the targets, they can either stimulate translation (as for DsrA and RprA) or cause message degradation. One novel small RNA, 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. When the target message is degraded, so is RyhB; both molecules require RNaseE for degradation. This coupled degradation appears to be true for other small RNAs as well. 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.