Effects of the nucleoid protein H-NS on RNAP elongation

描述（由申请人提供）：细菌染色体通过类核蛋白的压实和组织而浓缩超过1000倍[3,4]。热稳定类核结构蛋白（H-NS）是类核蛋白家族中研究最多的成员之一。H-NS与富含A/T的序列结合，可以通过寡聚化[11]形成大量DNA的核。由于启动子也具有较高的A/T含量，H-NS已被证明与启动子结合并抑制<5%的大肠杆菌基因[17]的转录。最近对H-NS的ChIP- ChIP研究表明，该蛋白可以与编码区高度转录的基因结合，这表明在转录过程中，伸长RNA聚合酶（RNAP）与H-NS相互作用。迄今为止，人们对H-NS相遇时对RNAP伸长的影响知之甚少。然而，在真核生物中，延长RNA pol II进入核小体的影响得到了很好的研究，核小体已被证明会导致RNA pol II回溯[19-25]。我的目标是揭示当一个延长的RNAP转录成H-NS结合的DNA时会发生什么。我们实验室最近的工作发现，隐式bglF操纵子有一个来自bglF基因的反义转录本，并且从反义启动子开始的转录可能直接进入H-NS补丁[2]。在我的第一个目标中，我计划识别并改变反义启动子。突变细胞系将通过2-葡萄糖苷酶测定来测试激活bgl操纵子的能力，以确定该反义启动子在调节bgl操纵子的激活中是否重要。在第二个目标中，我计划利用体外重建试验，在体外重现一个延长的RNAP转录成H-NS补丁。这种类型的分析已被用于测试延长RNA pol II转录到核小体中的体外效应[21,25]。一旦这个系统被优化，我就可以开始理解H-NS对RNAP的转录屏障机制。此外，我们可以在实验中添加延伸因子，以了解它们是帮助还是抑制RNAP通过H-NS结合的DNA转录。为了验证通过体外重构实验获得的数据，在第三个目标中，我计划使用ChIP-chip来了解RNAP在基因组水平上遇到类核蛋白的患病率。这项工作将开始揭示H-NS对延长RNAP的影响，以及它在调节大肠杆菌基因表达中的频率和意义。

DESCRIPTION (provided by applicant): The bacterial chromosome is condensed over 1000- fold through its compaction and organization by nucleoid proteins [3,4]. One of the most studied members of the nucleoid protein family is heat-stable nucleoid structuring protein (H-NS). H-NS binds to A/T rich sequences and can nucleate vast stretches of DNA through oligimerization [11]. Due to promoters having high A/T content as well, H-NS has been shown to bind to promoters and repress the transcription of <5% of E. coli genes [17]. Recent ChIP- chip studies of H-NS have shown that this protein can bind to highly transcribed genes in their coding regions, suggesting that elongating RNA polymerase (RNAP) interacts with H-NS during transcription [1]. To date, there is little known about the effects that H-NS has on an elongating RNAP when they encounter each other. However, in eukaryotes the effects of an elongating RNA pol II into nucleosomes are well studied, and nucleosomes have been shown to cause RNA pol II backtracking [19-25]. I aim to uncover what happens when an elongating RNAP transcribes into H-NS bound DNA. Recent work from our lab found that the cryptic bgl operon has an antisense transcript that originates from the bglF gene, and transcription that initiates from the antisense promoter presumably runs directly into an H-NS patch [2]. In my first aim I plan to identify and mutate the antisense promoter. Mutant cell lines will be tested for the ability activates the bgl operon through 2-glucosidase assays to see if this antisense promoter is important in regulating the activation of the bgl operon. In the second aim, I plan recapitulate an elongating RNAP transcribing into H-NS patches in vitro, using an in vitro reconstitution assay. This type of assay has been used to test the effects of an elongating RNA pol II transcribing into nucleosomes in vitro [21, 25]. Once this system has been optimized, I can start to understand the mechanism of the transcription barrier that H-NS has on RNAP. Additionally, we can add elongation factors to the assay to understand if they help or inhibit RNAP transcribe through H-NS bound DNA. To validate the data gained through the in vitro reconstitution assay, in the third aim I plan to use ChIP-chip to understand the prevalence of RNAP encountering nucleoid proteins at a genome level. This work will start to uncover the effects H-NS has on an elongating RNAP, and its frequency and significance in regulating gene expression in E. coli.