In Vivo Studies of Abortive Initiation

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单位RNA聚合酶(RNAP)进行的，从细菌到人类，RNAP在序列、结构和功能上都是保守的。虽然DNA聚合酶启动DNA合成需要使用一个引物，但人们普遍认为RNAP启动RNA合成是“从头开始”的(即RNAP仅使用游离的NTP启动RNA合成)。通过拟议的研究，我们试图挑战这一传统范式。具体地说，我们将调查这一假说，即很大一部分转录不是“从头开始”的，而是依赖于使用小的~2-5个NT RNA转录本“NanRNAs”，它通过作为启动转录的引物来影响基因的表达。第一个目标将使用微阵列分析、染色质免疫沉淀(CHIP)和完全平铺的高密度DNA微阵列(芯片上)，以及高通量测序来定义依赖于纳米RNA的启动如何影响基因表达。第二个目标将使用高通量测序来识别那些在体内启动转录启动功能的纳米RNA。第三个目标将使用微阵列分析、芯片上芯片和高通量测序来确定在生理生长条件下依赖于纳米RNA的启动对基因表达的影响程度。这项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，并同时发现一类新的调节性小RNA--“纳米RNAs”，它通过一种新的作用模式在所有活细胞中发挥作用。

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.