Effects of the nucleoid protein H-NS on RNAP elongation

核蛋白 H-NS 对 RNAP 延伸的影响

• 批准号: 8127032
• 负责人: Matthew V Kotlajich
• 金额: $ 4.84万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-04 至 2014-04-03
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8127032
• 关键词: Affect; Antibiotic Resistance; Area; Bacterial Chromosomes; Binding; Biological Assay; Cell Line; Cells; Code; Complex; DNA; DNA Binding; DNA Polymerase II; DNA-Binding Proteins; DNA-Directed RNA Polymerase; Data; Elongation Factor; Enterobacteriaceae; Escherichia coli; Eukaryota; Frequencies; Gene Expression; Genes; Genetic Transcription; Genome; Genomics; Glucosides; Health; Heating; Human; In Vitro; Mutate; Nucleic Acids; Nucleosomes; Operon; Prevalence; Promoter Regions; Protein Binding; Protein Family; Proteins; RNA; RNA Polymerase Inhibitor; Recombinants; Rifampin; Running; Site; Stretching; Structure; System; Testing; Transcript; Transcription Elongation; Transcription Initiation Site; Transcription Repressor/Corepressor; Transcriptional Regulation; Virulence; Work; cell type; combat; expectation; genome-wide; glucosidase; human PTCH2 protein; in vivo; knock-down; member; mutant; novel therapeutics; patched protein; prevent; promoter; protein structure; reconstitution; scaffold; sugar

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. PUBLIC HEALTH RELEVANCE: The nucleoid protein H-NS regulates the expression of numerous virulence genes along with horizontally acquired genes in enterobacteria, both of which cause significant threats to human health [36, 47, 50-54]. To date there is little known about how RNAP interacts with H-NS bound DNA. Understanding the effect that H-NS has on an elongating RNAP could provide a new therapeutic avenue to combat pathogenic enterobacteria or the spread of antibiotic resistance genes [54].

描述（由申请人提供）：细菌染色体通过类核蛋白的压缩和组织而浓缩超过1000倍[3，4]。类核蛋白家族中研究最多的成员之一是热稳定的类核结构蛋白（H-NS）。H-NS与富含A/T的序列结合，可以通过寡聚化使大量DNA片段成核[11]。由于启动子也具有高的A/T含量，H-NS已显示与启动子结合并抑制<5%的E. coli基因[17]。最近对H-NS的ChIP-芯片研究表明，该蛋白质可以在其编码区与高度转录的基因结合，这表明延伸RNA聚合酶（RNAP）在转录期间与H-NS相互作用[1]。到目前为止，很少有人知道的影响，H-NS对延长RNAP时，他们遇到对方。然而，在真核生物中，将RNA pol II延伸到核小体中的作用已得到充分研究，并且核小体已显示引起RNA pol II回溯[19-25]。我的目标是揭示当一个延伸的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，隐蔽的bgl操纵子具有源自bglF基因的反义转录物，并且从反义启动子启动的转录可能直接进入H-NS补丁[2]。在我的第一个目标中，我计划识别并突变反义启动子。将通过β-葡糖苷酶测定测试突变细胞系激活bgl操纵子的能力，以观察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，我计划使用体外重建试验，在体外重现转录成H-NS斑的延长RNAP。这种类型的测定已用于测试体外延长RNA pol II转录到核小体中的影响[21，25]。一旦这个系统被优化，我就可以开始理解H-NS对RNAP的转录障碍的机制。此外，我们可以将延伸因子添加到测定中，以了解它们是否有助于或抑制RNAP通过H-NS结合的DNA转录。为了验证通过体外重建试验获得的数据，在第三个目标中，我计划使用ChIP芯片来了解RNAP在基因组水平上遇到类核蛋白的流行程度。这项工作将开始揭示H-NS对延伸RNAP的影响，以及它在调节E.杆菌 公共卫生相关性：类核蛋白H-NS调节肠杆菌中许多毒力基因的表达，沿着水平获得的基因，这两者都对人类健康造成重大威胁[36，47，50-54]。迄今为止，人们对RNAP如何与H-NS结合的DNA相互作用知之甚少。了解H-NS对延长RNAP的影响可以提供一种新的治疗途径来对抗致病性肠杆菌或抗生素耐药基因的传播[54]。