Bacterial Functions Involved in Cell Growth Control

我们的研究集中在两种类型的转录后基因表达调节，即通过小调控RNA调节mRNA的降解和翻译，以及通过能量依赖的蛋白酶调节蛋白质的稳定性。Rpos是大肠杆菌中的一种中央应激反应调节因子，受到这两种水平的控制。RPOS的降解需要ClpXP蛋白酶和RSSB，RSSB是一种向蛋白酶递送RPOS的蛋白质。降解是通过RSSB的磷酸化来表示的；我们正在研究体内和体外的磷酸化和去磷酸化的机制。RSSB的缺失分析表明，C-末端的序列是底物正确释放到蛋白酶的关键；N-末端的序列是与蛋白酶相互作用所必需的。 RPOS翻译至少受两个小RNA的正向调控。RPOS翻译起始上游的信息折叠成一个发夹，阻断核糖体结合，从而阻止翻译。小的调控RNA，DSRA和RPRA，竞争发夹的抑制茎，破坏RPOS先导的二级结构，允许翻译。DsrA的启动子受温度调节(低温开启，高温关闭)。我们发现温度调节位于36个碱基对的最小启动子区域；虽然该区域的许多元件对温度调节起作用，但-10处的RNA聚合酶相互作用位点是最关键的。我们的结果表明，启动子结构的变化可能是温度调节的中介。RPRA被认为是dsrAmuants的多拷贝抑制因子，受RCSC和RCSB两个组分调节。这些调节剂还可以促进被膜多糖的合成和激活细胞分裂蛋白的合成；它们是由细胞表面压力激活的。当它们被激活时，RPOS合成以RPRA依赖的方式增加。似乎这种小的RNA在生物膜的形成过程中特别重要。 与Gisela Storz博士的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寻找新的小RNA的策略，并在最近找到了一类特定的小RNA，它与RNA伴侣Hfq结合。最初的搜索结果是鉴定出17个新的小RNA和6个新的小ORF。不同的小RNA在不同的生长条件下表达。Hfq是DSRA和RPRA作用所必需的一种RNA伴侣，被发现与大肠杆菌中约1/3的已知小RNA结合，并通过与Hfq的结合鉴定了20个新的小RNA。Hfq稳定小RNA，并帮助它们与mRNA靶标配对。一旦与目标配对，它们既可以刺激翻译(对于DSRA和RPRA)，也可以导致消息降级。对一种新的小RNA RyhB进行了较为详细的研究。我们发现它被毛皮抑制物抑制，因此当细胞内铁限制时，它会大量产生。当它被制造出来时，它针对多个mRNA进行降解。靶基因编码铁储存蛋白(铁蛋白)或含铁但非必需的代谢蛋白。因此，在弧菌、沙门氏菌、克雷伯氏菌和耶尔森氏菌中也发现了这种小RNA，它重新编程细胞中的铁利用，可能是一些病原体毒力的重要组成部分。当目标信息被降解时，RyhB也是如此；这两个分子都需要RNaseE来降解。这种伴随的降解似乎也适用于其他小RNA。另一种新颖的小RNA很可能代表同样有趣的新调控途径。此外，从我们在大肠杆菌中对小RNA的全基因组搜索中获得的信息正在形成将对小RNA的搜索扩展到其他细菌物种的基础。

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.