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。我们的实验室与其他人合作，进行了两次< 1 bb0 E的非编码rna的全球搜索。大肠杆菌</I>，对目前鉴定的80多种调控rna有重要贡献。大量这些RNA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，也在<I>弧菌</I中发现。沙门氏菌</I>，克雷伯氏菌</I>，耶尔森氏菌</I>，沙门氏菌</I>，沙门氏菌</I>，沙门氏菌</I>，耶尔森氏菌</I>，重新编程铁在细胞中的使用，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；这些小rna是作为OmpR/EnvZ调控的一部分在高渗透压下生成的，以前以其调节主要的外膜孔蛋白而闻名。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，并下调葡萄糖特异性转运蛋白mRNA的编码，该转运蛋白为<I>ptsG</I>。SgrS的诱导依赖于一种新的转录调节因子，由分化基因编码，我们将其命名为<I>sgrR</I>。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；这些目标与DsrA的目标不同。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，它们增加了<I>rpoS</I> mRNA的稳定性和翻译，保护其免受RNAse e的降解。所有这些hfq结合的sRNAs都与靶mRNA配对，但配对区域通常很短，中断，因此难以识别。此外，即使配对可以预测，调控也并不总是可见的。对OmrA/B RNA配对细节的检查表明，RNA的5端是与所有靶标配对所必需的。5端的灵活性可能允许更有效的配对。在其他实验中，我们选择了没有已知小RNA调节因子的基因，创建了与它们的翻译融合，并使用遗传筛选来识别sRNA翻译调节因子。我们的工作，结合其他实验室对这一调控家族的研究，表明细菌中的大量基因将受到这种转录后调控的影响。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。通过对RpoS降解调节因子的基因筛选，发现了一种以前未被表征的小蛋白YaiB，现在更名为IraP。<I>iraP</I>突变体在一定程度上降低了正常生长条件下RpoS的稳定性，完全破坏了磷酸盐饥饿后RpoS的稳定性。在纯化的体外系统中，IraP阻断RpoS的转换，并直接与RssB相互作用。在<我> E。在大肠杆菌</I b>中，磷酸盐饥饿通过小分子ppGpp水平的增加而被感知，并且ppGpp正调控<I>iraP</I>启动子。在沙门氏菌中，这种基因的表达也会在对镁缺乏的反应中被诱导。这种抗适配器只是这些蛋白质中的第一种。我们现在已经确定了另外两个小蛋白，YcgW（重命名为IraM）和YjiD（重命名为IraD）也可以在纯化的体外系统中稳定RpoS。IraM是对镁缺乏的反应；IraD在DNA损伤后很重要。至少有一个其他的反适配器可能存在，因为当所有已知的反适配器都是突变时，我们有条件导致RpoS稳定。因此，抗接头定义了一个新的调控水平，与RssB接头蛋白相互作用并阻断其作用能力；环境信号通过调节不同抗适配器的表达来调节RpoS的周转

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 collaboration with others, have undertaken two global searches for non-coding RNAs in <I>E. coli</I>, contributing significantly to the more than 80 regulatory RNAs that are now identified. A large number 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 <I> Vibrio</I., <I>Salmonella</I>, <I>Klebsiella</I>, and <I>Yersinia</I>, 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 as part of the OmpR/EnvZ regulon, previously known for its regulation of major outer membrane porins. 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 <I>ptsG</I>. SgrS induction depends on a novel transcriptional regulator, encoded by the divergent gene, named by us <I>sgrR</I>. 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; these targets differ from those 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 increase both the stability and translation of <I>rpoS</I> mRNA, protecting it from degradation by RNAse E. All of these Hfq-binding sRNAs pair with target mRNAs, but the regions of pairing are often short, interrupted, and therefore difficult to identify. In addition, even when pairing can be predicted, regulation is not always seen. Examination of the details of pairing by the OmrA/B RNAs demonstrate that the 5 end of the RNA is required for pairing to all targets. It is possible that flexibility at the 5 end allows more efficient pairing. In other experiments, we have chosen genes with no known small RNA regulators, created translational fusions to them, and used genetic screens to identify sRNA translational regulators. Our work, combined with work from other labs on this family of regulators, suggests that a large number of genes in bacteria will be subject to this post-transcriptional regulation. 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. A genetic screen for regulators of RpoS degradation led to discovery of a small, previously uncharacterized protein, YaiB, now renamed IraP. Mutants of <I>iraP</I> have somewhat decreased stability of RpoS under normal growth conditions and totally abolish the stabilization of RpoS after phosphate starvation. IraP blocks RpoS turnover in a purified in vitro system, and directly interacts with RssB. In <I>E. coli</I>, phosphate starvation is sensed by an increase in the levels of the small molecule ppGpp, and the <I>iraP</I> promoter is positively regulated by ppGpp. In Salmonella, expression of this gene is also induced in response to starvation for magnesium. This anti-adaptor is only the first of these proteins. We have now identified two other small proteins that also act to stabilize RpoS in a purified in vitro system, YcgW (renamed IraM), and YjiD (renamed IraD). IraM is made in response to magnesium starvation; IraD is important after DNA damage. At least one other anti-adaptor is likely to exist, since we have conditions that lead to RpoS stabilization when all the known anti-adaptors are mutant. Thus, the anti-adaptors define a new level of regulatory control, interacting with the RssB adaptor protein and blocking its ability to act; environmental signals regulate RpoS turnover by regulating expression of different antiadaptors