The Role Of Alternate Sigma Factors In The Transmission

替代 Sigma 因子在传输中的作用

• 批准号: 7196694
• 负责人: Frank Gherardini
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7196694
• 关键词: Role; Alternate; Sigma; Factors; Transmission

Regulation of gene expression by sigmaS, a member of the sigma70 family, is responsible for the transcription of a variety of genes in E. coli that are expressed as the cells enter stationary phase or during certain types of starvation and stress, such as acid stress or hyperosmotic stress. A role for sigmaS in the regulation of virulence factors has also been established in Salmonella sp. Salmonella plasmid virulence genes, spvR and spvABCD are carried on large (50 to 100-kb) plasmids in a variety of Salmonella species including S. typhimurium, S. choleraesuis, and S. dublin. Loss of these plasmids, of SpvR, SpvC and SpvD, or of sigmaS, results in loss of virulence in mouse models and the ability of the cells to multiply in the reticuloendothelial system. Expression of the genes increases dramatically as cells enter station phase and is dependent on sigmaS. The current model proposes that accumulation of sigmaS in response to growth phase, stress, or starvation conditions increases transcription of spvR, and sigmaS and SpvR then act together to increase transcription of the spv operon. Although additional regulatory factors often function to modulate the expression of specific sigmaS-dependent genes, the expression of these genes results largely from increased levels of sigmaS in the cell. In E. coli, sigmaS levels are controlled in a variety of ways, including transcription initiation and translational elongation. Some of the major factors that influence sigmaS levels, however, are post-translational events that lead to increased stability of the protein. During exponential growth at 37oC,sigmaS has a half-life of less than 2 min. As the cells enter stationary phase or encounter certain stress conditions, the half-life of sigmaS increases to greater than 30 min. The ClpXP protease is a cytoplasmic, ATP-dependent protease that is responsible for the rapid degradation of sigmaS during exponential growth. This rapid degradation of sigmaS by ClpXP requires RssB (also called SprE), a protein that shares homology with the family of response regulators and appears to specifically modulate the activity of sigmaS as well as its degradation. The heat shock protein DnaK has also been shown to have a positive role in the post-translational control of sigmaS. DnaK appears to be involved in the transduction of at least two signals, heat shock and carbon starvation, that result in reduced sigmaS turnover. In bacteria, sigmaS-RNA polymerase holoenzyme is responsible for gene expression following the transition from exponential growth to stationary phase or in response to certain types of stress. B. burgdorferi contains rpoS (encoding sigmaS) and our preliminary data indicate that expression of rpoS increases as cultures enter stationary phase. We have mapped a transcriptional start site of rpoS from B. burgdorferi to a potential sigma54-dependent promoter. Furthermore, analysis of the transcription of rpoS in a B. burgdorferi sigma54 mutant indicates that sigmaS is regulated by sigma54 as cells enter stationary phase of growth. Thus sigma54-holoenzyme is required for sigmaS expression and both proteins play key roles in the survival of B. burgdorferi in the tick midgut and in survival in mammalian hosts. (3) Regulation of gene expression by sigma54. As indicated above, we have identified a potential sigma54-dependent promoter that is involved in expression of rpoS. Sigma54, encoded by the rpoN gene, as first shown to be required for the expression of genes involved in nitrogen metabolism in enteric bacteria. It has since been shown to be required in various bacteria for the transcription of genes whose products are involved in such diverse functions as hydrogen metabolism, C4-dicarboxylic acid transport, pilin and flagellar biosynthesis, and degradation of aromatic compounds. Unlike other alternative sigma factors, sigma54 does not share homology with the sigma70 family. Moreover, the mechanism by which sigma54-RNA polymerase holoenyzme (sigma54-holoenzyme) initiates transcription differs from that of other forms of RNA polymerase holoenzyme. sigma54-Holoenzyme recognizes promoters that have conserved elements in the ?12 and ?24 regions, having the consensus sequence 5?-TGGCACN4TTTGC(A/T)-3?. The spacing between the conserved GG and GC doublets (underlined in the consensus sequence) is critical, as any changes in the spacing result in failure of sigma54-holoenzyme to recognize the promoter. Sigma54-Holoenzyme binds to the promoter to form a closed promoter complex, but it is unable to initiate transcription in the absence of an activator protein. The activator binds to sites that are usually located 100-200 bp upstream of the transcriptional start site and makes transient contact with sigma54-holoenyzme through DNA looping. Productive interactions between the activator and sigma54-holoenzyme lead to the isomerization of the closed promoter complex to an open complex that is transcriptionally competent. To catalyze this isomerization reaction, the activator must hydrolyze ATP. The mechanism by which the activator couples ATP hydrolysis to open complex formation is not known, but it does not appear to involve phosphorylation of either the activator or sigma54-holoenzyme. The activities of activators of sigma54-holoenzyme are regulated in response to environmental signals. Many of the activators of sigma54-holoenzyme are response regulators in two-component regulatory systems, and phosphorylation of these proteins results in their activation. These response regulators are phosphorylated by their cognate protein histidine kinases in response to an environmental signal. Once phosphorylated, the response regulator activates transcription of other genes. The activator of sigma54-holoenzyme from B. burgdorferi is also a response regulator of a two-component system. The gene encoding the activator is in an operon with a gene encoding its cognate protein histidine kinase. Our preliminary data indicate that the activator controls expression of rpoS. Therefore, we refer to this activator as sigmaS regulator (SisR) and its cognate protein histidine kinase as sigmaS regulatory protein histidine kinase (SisK).

sigmaS（sigma70 家族成员）对基因表达的调节负责大肠杆菌中多种基因的转录，这些基因在细胞进入静止期或某些类型的饥饿和应激（例如酸应激）期间表达或高渗应激。 sigmaS 在沙门氏菌毒力因子调节中的作用也已得到证实。沙门氏菌质粒毒力基因 spvR 和 spvABCD 携带在多种沙门氏菌属的大（50 至 100 kb）质粒上，包括鼠伤寒沙门氏菌、猪霍乱沙门氏菌和都柏林沙门氏菌。这些质粒、SpvR、SpvC 和 SpvD 或 sigmaS 的丢失，会导致小鼠模型中毒力的丧失以及细胞在网状内皮系统中繁殖的能力的丧失。当细胞进入静止期时，基因的表达急剧增加，并且依赖于 sigmaS。目前的模型提出，响应生长期、应激或饥饿条件而积累的 sigmaS 会增加 spvR 的转录，然后 sigmaS 和 SpvR 共同作用以增加 spv 操纵子的转录。尽管额外的调节因子通常起到调节特定 sigmaS 依赖性基因的表达的作用，但这些基因的表达很大程度上是由细胞中 sigmaS 水平增加引起的。在大肠杆菌中，sigmaS 水平通过多种方式控制，包括转录起始和翻译延伸。然而，影响 sigmaS 水平的一些主要因素是导致蛋白质稳定性增加的翻译后事件。在 37oC 指数生长期间，sigmaS 的半衰期小于 2 分钟。当细胞进入稳定期或遇到某些应激条件时，sigmaS 的半衰期增加至超过 30 分钟。 ClpXP 蛋白酶是一种细胞质、ATP 依赖性蛋白酶，负责在指数生长过程中 sigmaS 的快速降解。 ClpXP 对 sigmaS 的快速降解需要 RssB（也称为 SprE），这是一种与反应调节因子家族具有同源性的蛋白质，似乎可以特异性调节 sigmaS 的活性及其降解。热休克蛋白 DnaK 也被证明在 sigmaS 的翻译后控制中具有积极作用。 DnaK 似乎参与至少两个信号的转导，即热休克和碳饥饿，从而导致 sigmaS 周转减少。在细菌中，sigmaS-RNA 聚合酶全酶负责从指数生长期过渡到稳定期或响应某些类型的应激后的基因表达。伯氏疏螺旋体含有 rpoS（编码 sigmaS），我们的初步数据表明，随着培养物进入稳定期，rpoS 的表达增加。我们已经将伯氏疏螺旋体的 rpoS 转录起始位点定位到潜在的 sigma54 依赖性启动子。此外，对伯氏疏螺旋体 sigma54 突变体中 rpoS 转录的分析表明，当细胞进入生长稳定期时，sigmaS 受到 sigma54 的调节。因此，sigma54-全酶是 sigmaS 表达所必需的，并且这两种蛋白对于伯氏疏螺旋体在蜱中肠中的生存以及在哺乳动物宿主中的生存都起着关键作用。 (3)sigma54对基因表达的调节。如上所述，我们已经鉴定了一个参与 rpoS 表达的潜在 sigma54 依赖性启动子。 Sigma54，由 rpoN 基因编码，首次被证明是肠道细菌中氮代谢相关基因表达所必需的。此后，它已被证明是各种细菌基因转录所必需的，这些基因的产物涉及多种功能，如氢代谢、C4-二羧酸运输、菌毛蛋白和鞭毛生物合成以及芳香族化合物的降解。与其他替代 sigma 因子不同，sigma54 与 sigma70 家族不具有同源性。此外，sigma54-RNA聚合酶全酶（sigma54-holoenzyme）启动转录的机制与其他形式的RNA聚合酶全酶不同。 sigma54-Holoenzyme识别在α12和α24区具有保守元件的启动子，具有共有序列5′-TGGCACN4TTTGC(A/T)-3′。保守的 GG 和 GC 双联体（共有序列中下划线）之间的间距至关重要，因为间距的任何变化都会导致 sigma54-全酶无法识别启动子。 Sigma54-Holoenzyme 与启动子结合形成封闭的启动子复合物，但在没有激活蛋白的情况下无法启动转录。激活剂与通常位于转录起始位点上游 100-200 bp 的位点结合，并通过 DNA 环与 sigma54-holoenyzme 短暂接触。激活剂和 sigma54-全酶之间的有效相互作用导致封闭启动子复合物异构化为具有转录能力的开放复合物。为了催化这种异构化反应，活化剂必须水解 ATP。激活剂耦合 ATP 水解以开放复合物形成的机制尚不清楚，但它似乎不涉及激活剂或 sigma54-全酶的磷酸化。 sigma54-全酶激活剂的活性根据环境信号进行调节。 sigma54-全酶的许多激活剂是双组分调节系统中的反应调节剂，这些蛋白质的磷酸化导致它们的激活。这些反应调节剂响应环境信号而被其同源蛋白组氨酸激酶磷酸化。一旦磷酸化，反应调节器就会激活其他基因的转录。伯氏疏螺旋体的 sigma54-全酶激活剂也是双组分系统的反应调节剂。编码激活剂的基因位于操纵子中，该操纵子具有编码其同源蛋白组氨酸激酶的基因。我们的初步数据表明激活剂控制 rpoS 的表达。因此，我们将该激活剂称为 sigmaS 调节蛋白 (SisR)，将其同源蛋白组氨酸激酶称为 sigmaS 调节蛋白组氨酸激酶 (SisK)。