In vivo observations of transcription at single-nucleotide resolution

众所周知，RNA聚合酶（RNAP）的转录本延伸是一个不连续的过程：活跃的RNA合成周期经常被暂停所打断，在此期间，RNAP在恢复正常延伸之前暂时停止在DNA的特定位置。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中都是非常保守的，并且被认为是多种细胞过程的关键步骤。除了影响RNA生产的总体速度外，这些停顿还促进了外部调控因子的招募，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。迄今为止，细菌中转录暂停的实验主要局限于体外研究。然而，这些停顿是否在细胞环境中保持不变仍有待确定，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鸿沟，以评估暂停在整个细菌基因组中的流行程度，并确定它们在调节基因表达中的作用。

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.