In Vivo Studies of Abortive Initiation

流产起始的体内研究

• 批准号: 8444463
• 负责人: Bryce Edward Nickels
• 金额: $ 37.52万
• 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 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8444463
• 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; 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。所有细胞中的转录都是由多亚基RNA聚合酶（RNAP）进行的，这些RNA聚合酶在序列、结构和功能上从细菌到人类都是保守的。尽管通过DNA聚合酶启动DNA合成需要使用引物，但广泛接受的是通过RNAP启动RNA合成“从头”发生（即RNAP仅使用游离NTP启动RNA合成）。通过拟议的研究，我们试图挑战这种传统的范式。具体而言，我们将研究这样的假设，即转录的显著部分不会“从头”发生，而是依赖于使用小的~2-5 nt RNA转录物，“nanoRNA”，其通过充当引物来启动转录而影响基因表达。 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）结合，以及高通量测序来定义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nanoRNA。第三个目标将使用微阵列分析，ChIP芯片和高通量测序来确定nanoRNA依赖性引发在生理生长条件下影响基因表达的程度。 这项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，并同时发现一类新的调节性小RNA（“nanoRNA”），它们通过一种新的作用模式在所有活细胞中发挥作用。