In vivo observations of transcription at single-nucleotide resolution

单核苷酸分辨率转录的体内观察

• 批准号: 8414522
• 负责人: Matthew Herbert Larson
• 金额: $ 5.22万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-02-01 至 2015-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8414522
• 关键词: Affect; Antibiotics; Automobile Driving; Bacteria; Bacterial Genome; Bacterial RNA; Behavior; Biochemical; Bioinformatics; Biological Assay; Cancer Etiology; Cell physiology; Cells; Chlamydia trachomatis; Coupling; DNA; DNA Library; DNA-Directed RNA Polymerase; Data; Defect; Environment; Escherichia coli; Event; Frequencies; Gene Activation; Gene Expression; Gene Expression Regulation; Gene Silencing; Genes; Genetic; Genetic Transcription; Genetic Translation; Genome; Goals; Helicobacter pylori; Human; In Vitro; Lead; Life; Link; Malignant Neoplasms; Maps; Measurement; Mediating; Messenger RNA; Methodology; Methods; Modeling; Molecular; Monitor; Mutation; Nucleotides; Organism; Play; Positioning Attribute; Prevalence; Process; Production; Proteins; Protocols documentation; RNA; RNA Processing; RNA Splicing; RNA chemical synthesis; Resolution; Resources; Ribosomes; Role; Site; Structure; Techniques; Testing; Transcript; Translation Initiation; Translations; Tumor Suppression; Wit; Yeasts; cancer cell; carcinogenesis; deep sequencing; genome-wide; in vivo; insight; mutant; novel; pathogenic bacteria; protein complex; research study; tool; transcription factor; tumor growth

DESCRIPTION (provided by applicant): It is well established that transcript elongation by RNA polymerase (RNAP) is a discontinuous process: periods of active RNA synthesis are frequently interrupted by pauses during which RNAP momentarily halts at specific positions along the DNA before resuming normal elongation. Transcriptional pausing by multi-subunit RNAP molecules is remarkably conserved across different organisms, from cancer-associated bacteria to humans, and has been implicated as a key step in a variety of cellular processes. In addition to affecting the overall rate of RNA production, it has been proposed that these pauses facilitate the recruitment of external regulatory factors, the synchronization of transcription wit translation, and the promotion of a variety of RNA processing events, including cotranscriptional folding, splicing, and termination. To date, experiments on transcriptional pausing in bacteria have largely been restricted to in vitro studies. However, it remains to be established whether these pauses persist unchanged in the cellular environment, where both ribosomes and transcription factors may alter transcription dynamics. In this study we aim to bridge the divide between in vitro and in vivo transcription measurements in order to assess the prevalence of pausing across the complete bacterial genome, as well as to determine their role in regulating gene expression. The ability to globally monitor both transcription and translation in vivo was recently pioneered by the Weissman lab. Originally demonstrated in yeast, they showed that RNAP- or ribosome-associated transcripts could be rapidly isolated from live cells, converted into a DNA library without introducing significant bias, and ultimately quantified using massively parallel deep-sequencing techniques. This methodology allows for the identification of transcriptional and translational pause sites across the entire genome with single-nucleotide resolution, and represented a significant advance over other in vivo tracking techniques that suffered from limited spatial and temporal resolution. I aim to further develop high-resolution RNAP profiling by creating a comparable assay capable of monitoring transcription in E. coli. By comparing this transcriptional profiling pause data with previous in vitro studies, I can build a comprehensive top-down model of transcriptional pausing that explains both the molecular mechanism by which RNAP pauses, as well as the function of these pauses in live cells. These maps of RNAP pausing in WT E. coli will also be compared with mutant strains in which RNAP/ribosome coupling is compromised, either through mutations to the ribosome or to transcription factors thought to physically link transcription with translation. The methodologies developed will provide insight into the proliferation of pathogenic bacteria linked with carcinogenesis, such as Helicobacter pylori (H. pylori) and Chlamydia trachomatis (C. trachomatis), as well as providing a useful tool to probe the role transcriptional pausing in human cancer cells.

描述(申请人提供)：众所周知，RNA聚合酶(RNAP)的转录延长是一个不连续的过程：活跃的RNA合成周期经常被暂停中断，在此期间，RNAP在DNA上的特定位置短暂停止，然后恢复正常的延长。多亚基RNAP分子的转录暂停在从癌症相关细菌到人类的不同生物体中都非常保守，并被认为是各种细胞过程中的关键步骤。除了影响RNA的总体生产速度外，有人认为这些暂停还有助于招募外部调节因子，促进转录与翻译的同步，以及促进各种RNA加工事件，包括共转录折叠、剪接和终止。到目前为止，关于细菌转录暂停的实验在很大程度上局限于体外研究。然而，这些停顿是否在细胞环境中保持不变仍有待确定，在细胞环境中，核糖体和转录因子都可能改变转录动力学。在这项研究中，我们的目标是弥合体外和体内转录测量之间的分歧，以评估整个细菌基因组中暂停的发生率，以及确定它们在调控基因表达中的作用。全球监测体内转录和翻译的能力最近由魏斯曼实验室率先开发。他们最初在酵母中展示了RNAP或核糖体相关的转录本可以从活细胞中快速分离出来，转化成DNA文库，而不会产生显著的偏差，并最终使用大规模平行的深度测序技术进行定量。这种方法允许以单核苷酸分辨率识别整个基因组的转录和翻译暂停位置，与其他受空间和时间分辨率限制的体内跟踪技术相比是一个重大进步。我的目标是进一步开发高分辨率的RNAP图谱，通过创建一种能够监测大肠杆菌转录的类似方法。通过将这种转录暂停数据与之前的体外研究进行比较，我可以建立一个全面的自上而下的转录暂停模型，解释RNAP暂停的分子机制，以及这些暂停在活细胞中的功能。这些RNAP在WT大肠杆菌中暂停的图谱也将与RNAP/核糖体偶联受到损害的突变菌株进行比较，要么是通过核糖体的突变，要么是通过被认为在物理上将转录与翻译联系起来的转录因子。所开发的方法学将深入了解与致癌有关的病原菌的增殖，如幽门螺杆菌(H.Pylori)和沙眼衣原体(Ctrachomatis)，并为探索转录暂停在人类癌细胞中的作用提供有用的工具。