In vivo observations of transcription at single-nucleotide resolution

单核苷酸分辨率转录的体内观察

基本信息

批准号：
8254070
负责人：
Matthew Herbert Larson
金额：
$ 4.92万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
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/8254070
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 genome-wide in vivo insight mutant novel pathogenic bacteria protein complex research study tool transcription factor tumor growth

项目摘要

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.

众所周知，RNA聚合酶（RNAP）的转录本伸长率是一个不连续的过程：活动RNA合成的周期经常被停顿中断，在此期间，RNAP在恢复正常延长之前暂时停止沿着DNA的特定位置。从与癌症相关的细菌到人类，多生RNAP分子通过多生RNAP分子暂停的转录暂停，并且已被视为各种细胞过程的关键步骤。除了影响RNA产生的总体速度外，还提出这些暂停有助于募集外部调节因素，转录机智翻译的同步以及促进各种RNA处理事件，包括折叠折叠，旋转，旋转，旋转，旋转，终止和终止。迄今为止，对细菌中转录暂停的实验在很大程度上仅限于体外研究。但是，是否在细胞环境中持续不变，在核糖体和转录因子可能会改变转录动力学的情况下，这些暂停是否持续不变，还有待确定。在这项研究中，我们旨在弥合体外和体内转录测量之间的鸿沟，以评估整个细菌基因组中暂停的流行，并确定它们在调节基因表达中的作用。魏斯曼实验室最近率先启用了全球监测转录和翻译的能力。他们最初在酵母中证明，它们表明可以从活细胞中快速分离RNAP或核糖体相关的转录本，并转化为DNA文库而不引入明显的偏见，并最终使用大量平行的深层序列技术进行定量。该方法可以通过单核苷酸分辨率鉴定整个基因组中的转录和翻译暂停位点，并代表了与其他空间和时间分辨率有限的体内体内跟踪技术相比，它的显着进步。我的目标是通过创建能够监测大肠杆菌转录的可比测定法，进一步开发高分辨率的RNAP分析。通过将这种转录分析的暂停数据与以前的体外研究进行比较，我可以建立一个全面的转录暂停模型，这既解释了RNAP暂停的分子机制，又解释了这些暂停在活细胞中的功能。这些在WT大肠杆菌中暂停的RNAP暂停图也将与突变菌株进行比较，其中RNAP/核糖体偶联是通过突变与核糖体的突变或转录因子被损害的，或者是转录因子，被认为是从物理上将转录与翻译联系起来的。所开发的方法将洞悉与癌变有关的致病细菌的增殖，例如幽门螺杆菌（H. Pylori）和沙眼衣原体（沙眼曲霉），以及提供有用的工具，可探测在人类癌细胞中疾病转录的有用工具。