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).

Sigma70家族的成员Sigmas对基因表达的调节负责大肠杆菌中各种基因的转录，这些基因表示为细胞进入固定期或某些类型的饥饿和压力，例如酸应激或高渗透应激。在沙门氏菌SP中，也已经确定了Sigmas在调节毒力因子中的作用。沙门氏菌质粒毒力基因，SPVR和SPVABCD在多种沙门氏菌物种中携带在大型（50至100-kb）的质粒上，包括鼠伤寒沙门氏菌，霍乱链球菌和都柏林链球菌。这些质粒的损失，SPVR，SPVC和SPVD或Sigmas的损失导致小鼠模型中的毒力损失以及细胞在网状内皮系统中繁殖的能力。随着细胞进入站相位，基因的表达急剧增加，并取决于sigmas。当前的模型提出，响应生长阶段，应力或饥饿条件的sigmus积累会增加SPVR的转录，然后Sigmas和SPVR共同起作用以增加SPV操纵子的转录。尽管其他调节因子通常发挥作用以调节特定辅助依赖性基因的表达，但这些基因的表达在很大程度上是由于细胞中的Sigmas水平增加而导致的。在大肠杆菌中，西格玛水平通过多种方式控制，包括转录启动和翻译伸长。但是，影响闪光水平的一些主要因素是翻译后事件，导致蛋白质的稳定性提高。在37oC的指数增长过程中，Sigmas的半衰期不到2分钟。随着细胞进入固定相或遇到某些应力条件，Sigmam的半衰期增加到30分钟以上。 CLPXP蛋白酶是一种细胞质，依赖ATP的蛋白酶，导致指数生长过程中sigmas的迅速降解。 CLPXP对SIGMAS的这种快速降解需要RSSB（也称为SPRE），该蛋白质与响应调节剂家族共享同源性，并且似乎特别调节了Sigmas的活性及其降解。热休克蛋白DNAK也已显示在散射后控制中具有积极作用。 DNAK似乎参与了至少两个信号，热冲击和碳饥饿，从而导致Sigmus更换减少。在细菌中，Sigmas-RNA聚合酶全酶在从指数生长到固定相或对某些类型的应激的响应后转变后负责基因表达。 B. burgdorferi包含RPO（编码sigmas），我们的初步数据表明，随着培养物进入固定相，RPOS的表达增加。我们已将RPO的转录起始位点从B. burgdorferi绘制为潜在的SIGMA54依赖启动子。此外，对B. burgdorferi Sigma54突变体中RPO的转录的分析表明，随着细胞进入生长的固定相，Sigma54对Sigmam进行调节。因此，Sigma54- holo酶是Sigmas表达所必需的，并且两种蛋白质在B. burgdorferi在Tick Midgut的生存中起着关键作用，并且在哺乳动物宿主中的生存中起着关键作用。 （3）SIGMA54调节基因表达。如上所述，我们已经确定了参与RPOS表达的潜在SigMA54依赖性启动子。由RPON基因编码的Sigma54首先证明是肠道细菌中参与氮代谢的基因表达所必需的。此后，在各种细菌中都需要进行它的转录，其产物参与氢代谢，C4-二羧酸转运，PILIN和鞭毛生物合成以及芳族化合物的降解等多种功能。与其他替代Sigma因素不同，Sigma54与Sigma70家族没有同源性。此外，SIGMA54-RNA聚合酶Holoenyzme（Sigma54- holoenzyme）启动转录与其他形式的RNA聚合酶全酶的不同。 Sigma54- holoenzyme识别在12和24个区域中具有保守元素的启动子，并具有共识序列5？-TGGCACN4TTTTGC（A/T）-3？？保守的GG和GC双峰（共识序列下划线）之间的间距至关重要，因为间距的任何变化都会导致Sigma54- holoenzyme识别启动子的任何变化导致sigma54- holoenzyme失败。 Sigma54-氢酶与启动子结合以形成封闭的启动子络合物，但在没有激活蛋白的情况下，它无法启动转录。激活因子与通常位于转录起始位点上游的100-200 bp的位点结合，并通过DNA循环与Sigma54-Holoenyzme进行短暂接触。激活剂和Sigma54-氢酶之间的生产性相互作用导致封闭启动子复合物的异构化与转录合理的开放配合物。为了催化这种异构化反应，激活剂必须水解ATP。激活剂夫妻ATP水解以打开复合形成的机制尚不清楚，但似乎不涉及激活剂或Sigma54- holoenzyme的磷酸化。 Sigma54-氢酶的活化剂的活性受环境信号的响应。 Sigma54-氢酶的许多激活剂是两个组分调节系统中的响应调节剂，这些蛋白质的磷酸化导致它们的激活。这些响应调节剂的同源蛋白组氨酸激酶会响应环境信号而磷酸化。一旦磷酸化，响应调节剂就会激活其他基因的转录。 B. burgdorferi的Sigma54-氢酶的激活剂也是两个组件系统的响应调节剂。编码活化剂的基因是在具有编码其同源蛋白组氨酸激酶的基因的操纵子中。我们的初步数据表明激活剂控制RPO的表达。因此，我们将此激活剂称为sigmas调节剂（SISR）及其同源蛋白组氨酸激酶作为sigmas调节蛋白组氨酸激酶（SISK）。