Bacterial Functions Involved in Cell Growth Control

近十年来，小分子非编码rna在调控中的重要作用已被认识并开始研究。我们的实验室与许多小组合作，在大肠杆菌中进行了两次全球非编码rna的搜索，对现在确定的80多个非编码rna做出了重大贡献。通过免疫沉淀和微阵列分析，我们发现这些RNA中至少有三分之一与RNA伴侣Hfq紧密结合。我们和其他人已经证明，每个与Hfq紧密结合的RNA都通过与靶mRNA配对来调节mRNA的稳定性和翻译，无论是积极的还是消极的。我们的实验室已经详细研究了许多这样的小rna。我们发现，每一种小RNA的表达都受到不同应激条件的调控，小RNA在适应应激中起着重要的作用。其中研究得最好的是RyhB。RyhB的转录受到依赖于铁的抑制因子的抑制，因此当细胞内铁受到限制时，小RNA就会大量产生。当它被制造出来时，它的目标是编码铁结合蛋白的mrna，以便降解。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，它可以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。另外两种小rna，现在被称为OmrA和OmrB，已被发现调节许多外膜蛋白；这些在高渗透压下生成的小rna是OmpR/EnvZ调控的一部分。对另一种hfq结合RNA RybB的调控研究表明，它依赖于另一种sigma因子sigma E进行转录。当错误折叠的外膜蛋白积聚在细胞的周质中时，Sigma E变得活跃。RybB可能在合成和活性水平上自动调节sigma E。RybB活性的证明表明，向外膜的运输可能比以前所知的更为严格。这些rna是一个不断增长的调节rna家族的特征，它们调节细胞表面，可能在感染期间很重要。另一种小RNA，现在被命名为SgrS，是当细胞积累葡萄糖-6-磷酸或磷酸化葡萄糖类似物时产生的，并下调mRNA的表达。葡萄糖特异性转运蛋白，由ptsG编码。SgrS的诱导依赖于一种新的转录调控因子，由分化基因编码，我们将其命名为sgrR。SgrR蛋白是转录调控因子保守家族中第一个被研究的成员（之前被错误注释），它直接结合DNA，负向自动调节，并可能直接感知磷酸糖的积累。当小RNA或转录调节因子突变时，细胞无法从葡萄糖-磷酸盐积累中恢复。先前的研究表明，DsrA和RprA两种小rna在正向调节应激sigma因子RpoS的翻译中发挥作用。最近，我们已经证明RprA也有许多其他的mRNA靶点，这些靶点是负调控的；这在之前的DsrA中已经被证明。新的RprA靶点扩大了RprA及其调控因子RcsC、RcsD和RcsB在控制该细菌生物膜形成中的可能作用。对DsrA和RprA作用机制的研究表明，它们既影响rpoS mRNA的稳定性，又影响rpoS mRNA的翻译。RpoS也受到蛋白质周转水平的控制。在活性生长过程中，RpoS会迅速降解，这一过程需要能量依赖性ClpXP蛋白酶和适配器蛋白RssB（一种可磷酸化的蛋白，可将RpoS呈现给蛋白酶）。RpoS经过各种应激或饥饿处理后趋于稳定；稳定模式一直是个谜。我们实验室和其他实验室最近的研究表明，在没有磷酸化的情况下，降解有显著的调节作用。

In the last decade, the important role of small non-coding RNAs in regulation have been recognized and begun to be studied. Our laboratory, in collaborations with a number of groups, have undertaken two global searches for non-coding RNAs in E. coli, contributing significantly to the more than 80 that are now identified. Using immunoprecipitation and analysis with microarrays, we find that at least one-third of these RNAs bind tightly to the RNA chaperone Hfq. We and others have shown that every RNA that binds tightly to Hfq acts by pairing with target mRNAs, regulating stability and translation of the mRNA, either positively or negatively. Our lab has studied a number of these small RNAs in detail. We have found that expression of each small RNA is regulated by different stress conditions, and that the small RNA plays an important role in adapting to stress. 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 mRNAs that encode iron-binding proteins for degradation. 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. Two other small RNAs, now called OmrA and OmrB, have been found to regulate a number of outer membrane proteins; these small RNAs are made at high osmolarity are part of the OmpR/EnvZ regulon. Studies on the regulation of RybB, another Hfq-binding RNA, have demonstrated that it is dependent on an alternative sigma factor, Sigma E, for transcription. Sigma E becomes active when misfolded outer membrane proteins accumulate in the periplasm of the cell. RybB autoregulates sigma E, possibly at both the level of synthesis and activity. The demonstration of the activity of RybB suggests that trafficking to the outer membrane may be even more tightly regulated than previously known. These RNAs are characteristic of a growing family of regulatory RNAs that regulate the cell surface, possibly important during infection. Another small RNA, now named SgrS, is made when cells accumulate glucose-6-phosphate or a phosphorylated glucose analog, and down-regulates the mRNA for the . glucose-specific transporter, encoded by ptsG. SgrS induction depends on a novel transcriptional regulator, encoded by the divergent gene, named by us sgrR. The SgrR protein, which is the first studied member of a conserved family of transcriptional regulators (previously mis-annotated), directly binds DNA, negatively autoregulates, and 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. Previous studies had demonstrated the roles of two small RNAs, DsrA and RprA, in positively regulating translation of the stress sigma factor RpoS. More recently, we have shown that RprA also has a number of other mRNA targets, which are negatively regulated; this had been previously shown for DsrA. The new RprA targets expand the likely role of RprA and its regulators, RcsC, RcsD, and RcsB, in controlling biofilm formation by this bacteria. Studies on the mechanism of action of DsrA and RprA suggest that they affect both the stability and translation of rpoS mRNA. RpoS is subject to control at the level of protein turnover as well. RpoS is rapidly degraded during active growth, in a process that requires the energy-dependent ClpXP protease and the adaptor protein RssB, a phosphorylatable protein that presents RpoS to the protease. RpoS becomes stable after various stress or starvation treatments; the mode of stabilization has been a mystery. Recent studies in our labs and others demonstrated significant regulation of degradation in the absence of phosphorylation.