Bacterial Functions Involved in Cell Growth Control

戈特斯曼 项目名称摘要 我们的研究重点是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 是大肠杆菌中的一个中心应激反应调节因子，受到这两个级别的控制。 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）。降解由 RssB 磷酸化发出；我们正在研究体内和体外磷酸化和去磷酸化的机制。然而，最近的研究表明，在没有磷酸化的情况下，降解具有显着的调节作用，这表明需要了解一种新的调节方法。 RssB 的删除分析表明 C 末端的序列对于底物正确释放到蛋白酶至关重要；与蛋白酶相互作用需要 N 端序列。 RpoS 翻译受到至少两个小 RNA 的正调控。 RpoS 翻译起始点上游的消息折叠成发夹，从而阻碍核糖体结合，从而阻碍翻译。小调节 RNA DsrA 和 RprA 竞争发夹的抑制茎，破坏 RpoS 前导序列的二级结构，从而实现翻译。 dsrA的启动子受温度调控；我们之前已经表明，调控存在于最小的启动子区域，其中 -10 区域发挥着关键作用。 RprA 被鉴定为 dsrAmutants 的多拷贝抑制子，受 RcsC、RcsD、RcsB 磷酸中继调控系统的调控。这些调节因子还可以促进荚膜多糖的合成，上调细胞分裂蛋白以及许多其他基因，其中一些基因参与生物膜的形成；它们被细胞表面压力激活。遗传学研究表明，感应激活磷酸继电器通常是通过修改 RcsF 的活性或结构来实现的，RcsF 是一种作用于膜传感器 RcsC 上游的脂蛋白。另外两种似乎调节 RpoS 的小 RNA 正在研究中。最后，我们发现了一些新的 RpoS 翻译调节水平，这些水平不依赖于小 RNA。 tmRNA（一种用于在停滞核糖体上标记肽以进行降解并缓解停滞的 RNA）和 miaA 指导的 RNA 修饰对于高水平的 RpoS 翻译都是必需的。因此，多种细胞系统都会影响 RpoS 水平。 与 NICHD 的 Gisela Storz 实验室合作，在全基因组范围内搜索保守的小 RNA。我们研究了在该搜索中发现的许多小 RNA 的作用和调控。其中研究最好的是 RyhB。 RyhB 转录受到 Fur（铁依赖性阻遏蛋白）的抑制，因此当细胞内铁有限时，小 RNA 会大量产生。当它被制造出来时，它会针对多个 mRNA 进行降解。目标 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，它会重新编程细胞中铁的使用，并且可能是某些病原体毒力的重要组成部分。利用 RyhB 的特性，我们与 NCI 的 David Fitzgerald 博士和 Peter Fitzgerald 博士以及科罗拉多州的 Michael Vasil 博士合作，证明了类似的小 RNA 调节铜绿假单胞菌中编码含铁蛋白的基因，尽管调节 RNA 的序列非常不同。在对 RyhB 的进一步研究中，我们证明 RyhB 及其靶标 mRNA 都会快速降解，这取决于必需的核酸内切酶 RNase E。只有当其他基因的转录活跃时，小 RNA 的降解才会快速，这表明与 mRNA 靶标配对对于小 RNA 的降解至关重要。基于RyhB的研究，我们能够证明这一类的另外两个小RNA，DsrA和OxyS，在转录活跃时也不稳定，但在转录不活跃时稳定；这可能是这些小 RNA 的一般特性。此次研究中发现的另外两种小 RNA RygA 和 RygB 可以调节许多外膜蛋白；这些小 RNA 是在渗透休克后产生的，似乎是 OmpR/EnvZ 调节子的一部分。 在与 Gisela Storz 博士实验室的持续合作中，我们利用 RNA 伴侣 Hfq 与许多小 RNA 紧密结合的能力来识别大肠杆菌中更多的小 RNA。对 Hfq 进行免疫沉淀，分离结合的 RNA 并用于探测微阵列。至少鉴定出另外 6 个小 RNA，另外可能有 6-10 个；全部都使用 Hfq，这表明它们通过与目标 mRNA 配对来发挥作用。其中一个最初被称为 RyaA，现在更名为 SgrS，已经得到了一些详细的研究。当细胞积累葡萄糖-6-磷酸或磷酸化葡萄糖类似物时，就会产生 SgrS。当葡萄糖被葡萄糖 PTS 转运蛋白转运到细胞内时，就会产生磷酸糖；葡萄糖特异性基因由 ptsG 编码。 H. Aiba 及其同事的工作表明，当进一步的代谢受阻时，磷酸糖的积累是有毒的，细胞会通过降解 ptsG 的 mRNA 来做出反应。我们发现这是由 SgrS 介导的，预计 SgrS 与 ptsG 5' UTR 配对。 SgrS 诱导依赖于一种新型转录调节因子，由不同基因 yabN 编码，我们将其重新命名为 sgrR 。 SgrR蛋白可以直接感知磷酸糖的积累。当小RNA或转录调节因子发生突变时，细胞无法从葡萄糖-磷酸盐积累中恢复。 DsrA 和 RprA 在 RpoS 翻译的正调节中的作用相当独特，并提出了配对是否只是简单地打开发夹或对靶 mRNA 和小 RNA 的加工或稳定性有影响的问题。使用先前测试的小RNA和目标信息中的突变和补偿突变来提供特异性，我们可以证明小RNA与rpoS信息配对会导致更高水平的信息，并且似乎也稳定了小RNA。对此的进一步研究应该会增加我们对小RNA积极作用的理解。

Gottesman Project Title Summary 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. However, recent studies demonstrated significant regulation of degradation in the absence of phosphorylation, suggesting a novel method of regulation that needs to be understood. 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 ; we have previously shown that regulation resides in a minimal promoter region, with the -10 region playing a critical role. RprA, identified as a multicopy suppressor of dsrAmutants, is regulated by the RcsC, RcsD, RcsB phosphorelay regulatory system. These regulators also act to turn up capsular polysaccharide synthesis and to up regulate a cell division protein as well as many other genes, some involved in biofilm formation; they are activated by cell surface stress. Genetic studies have demonstrated that sensing to activate the phosphorelay is usually via modification of the activity or structure of RcsF, a lipoprotein that acts upstream of the membrane sensor, RcsC. Two additional small RNAs that appear to regulate RpoS are under study. Finally, some novel levels of regulation of translation of RpoS have been found that are independent of small RNAs. Both tmRNA, an RNA that is used at stalled ribosomes to tag the peptide for degradation and relieve the stalling, and the miaA-directed RNA modification are necessary for high levels of RpoS translation. Thus, a variety of cellular systems affect RpoS levels. A genome-wide search for conserved small RNAs was carried out in collaboration with Gisela Storz's laboratory in NICHD. We have investigated the roles and regulation for a number of the small RNAs found in that search. The best-studied of these is RyhB. RyhB transcription is repressed by the Fur, iron-dependent repressor, and the small RNA 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. Using the properties of RyhB, we collaborated with Dr. David Fitzgerald and Dr. Peter Fitzgerald at NCI as well as Dr. Michael Vasil in Colorado to demonstrate that similar small RNAs regulate genes encoding iron-containing proteins in Pseudomonas aeruginosa, although the sequences of the regulatory RNAs are very different. In further studies on RyhB, we demonstrated that both RyhB and its target mRNAs are rapidly degraded, dependent upon an essential endonuclease, RNase E. Degradation of the small RNA was only rapid when transcription of other genes was active, suggesting that pairing with mRNA targets was critical for degradation of the small RNA. Based on the studies of RyhB, we were able to show that two other small RNAs of this class, DsrA and OxyS, were also unstable when transcription was active but stable when it was inactive; this is probably a general property of these small RNAs. Two other small RNAs from this search, RygA and RygB, have been found to regulate a number of outer membrane proteins; these small RNAs are made after osmotic shock and appear to be part of the OmpR/EnvZ regulon. In a continued collaboration with Dr. Gisela Storz's laboratory, we used the ability of the RNA chaperone Hfq to bind tightly to many small RNAs to identify yet more small RNAs in E. coli. Hfq was immunoprecipitated, the bound RNA isolated and used to probe microarrays. At least six additional small RNAs were identified with another 6-10 likely; all use Hfq, which suggests that they act by pairing with target mRNAs. One of these, originally called RyaA and now renamed SgrS, has been studied in some detail. SgrS is made when cells accumulate glucose-6-phosphate or a phosphorylated glucose analog. The sugar phosphate is made when glucose is transported into the cells by the glucose PTS transporter; the glucose-specific gene is encoded by ptsG. Work by H. Aiba and colleagues had demonstrated that when further metabolism is blocked, the accumulation of the sugar phosphate is toxic and cells respond by degrading the mRNA for ptsG. We find that this is mediated by SgrS, which is predicted to pair with the ptsG 5' UTR. SgrS induction depends on a novel transcriptional regulator, encoded by the divergent gene, yabN , renamed by us sgrR . The SgrR protein may directly sense the accumulation of sugar phosphate. When either the small RNA or the transcriptional regulator are mutant, cells are unable to recover from glucose-phosphate accumulation. The action of DsrA and RprA in positive regulation of RpoS translation is rather unique and raised the question whether pairing simply opens a hairpin or has effects on the processing or stability of the target mRNA and the small RNA. Using previously tested mutations and compensating mutations in the small RNAs and the target messages to provide specificity, we can show that small RNA pairing with the rpoS message results in higher levels of the message, and also seems to stabilize the small RNAs. Further studies on this should increase our understanding of positive action of small RNAs.