In Vivo Studies of Abortive Initiation

流产起始的体内研究

• 批准号: 8634118
• 负责人: Bryce Edward Nickels
• 金额: $ 38.9万
• 依托单位: RUTGERS, THE STATE UNIV OF N.J.
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-15 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8634118
• 关键词: Address; Affect; Bacteria; Cells; Collection; Coupled; DNA Microarray Chip; DNA biosynthesis; DNA-Directed DNA Polymerase; DNA-Directed RNA Polymerase; Development; Disease Pathway; Exonuclease; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Transcription; Goals; Gram-Negative Bacteria; Growth; Health; High-Throughput Nucleotide Sequencing; Homeostasis; Human; In Vitro; Lead; Life; Mediating; Microarray Analysis; Modeling; Phase; Physiological; Process; Pseudomonas aeruginosa; RNA; RNA chemical synthesis; Research; Small RNA; Structure; Transcript; Transcription Initiation; Work; base; chromatin immunoprecipitation; density; detector; in vivo; novel; oligoribonuclease; public health relevance

DESCRIPTION (provided by applicant): Transcription is the first step in gene expression and the step at which most gene regulation occurs. Transcription in all cells is carried out by multi-subunit RNA polymerases (RNAPs) that are conserved in sequence, structure and function from bacteria to humans. Whereas initiation of DNA synthesis by DNA polymerase requires use of a primer, it is widely accepted that the initiation of RNA synthesis by RNAP occurs "de novo" (i.e. RNAP initiates RNA synthesis using free NTPs only). With the proposed research we seek to challenge this conventional paradigm. Specifically, we will investigate the hypothesis that a significant fraction of transcription does not occur "de novo", but rather relies upon use of small ~2-5 nt RNA transcripts, "nanoRNAs", that influence gene expression by serving as primers to initiate transcription. The first aim will use microarray analysis, chromatin immunoprecipitation (ChIP) coupled with fully tiled high density DNA microarrays (ChIP-on-chip), and high-throughput sequencing to define how nanoRNA- dependent priming can influence gene expression. The second aim will use high-throughput sequencing to identify those nanoRNAs that function to prime transcription initiation in vivo. The third aim will use microarray analysis, ChIP-on-chip, and high-throughput sequencing to determine the extent to which nanoRNA-dependent priming impacts gene expression during physiological growth conditions. The proposed research is part a long-range effort to elucidate fundamental mechanisms of gene regulation in bacteria. These studies have the potential to redefine our view of a fundamental process that occurs in all living cells (i.e. transcription) and, in parallel, uncover a novel class of regulatory small RNAs, "nanoRNAs", that function in all living cells via a novel mode of action.

描述（由申请人提供）： 转录是基因表达的第一步，也是大多数基因调控发生的步骤。所有细胞中的转录均由多亚基 RNA 聚合酶 (RNAP) 进行，这些酶在序列、结构和功能上从细菌到人类都是保守的。尽管 DNA 聚合酶启动 DNA 合成需要使用引物，但人们普遍认为 RNAP 启动 RNA 合成是“从头”发生的（即 RNAP 仅使用游离 NTP 启动 RNA 合成）。通过所提出的研究，我们寻求挑战这种传统范式。具体来说，我们将研究这样一个假设，即很大一部分转录不是“从头”发生的，而是依赖于使用约 2-5 nt RNA 转录本（“nanoRNA”），这些转录本通过充当启动转录的引物来影响基因表达。 第一个目标将使用微阵列分析、染色质免疫沉淀 (ChIP) 与完全平铺的高密度 DNA 微阵列 (ChIP-on-chip) 相结合，以及高通量测序来确定 nanoRNA 依赖性引发如何影响基因表达。第二个目标将使用高通量测序来鉴定那些在体内启动转录起始的纳米RNA。第三个目标将使用微阵列分析、芯片上 ChIP 和高通量测序来确定生理生长条件下纳米 RNA 依赖性启动对基因表达的影响程度。 拟议的研究是阐明细菌基因调控基本机制的长期努力的一部分。这些研究有可能重新定义我们对所有活细胞中发生的基本过程（即转录）的看法，同时揭示一类新型的调节性小RNA，即“纳米RNA”，它通过一种新颖的作用模式在所有活细胞中发挥作用。