Mechanisms of transcription pausing and fidelity in prokaryotes and eukaryotes

转录保真度：在我们研究转录保真度的方法中，我们结合了为我们小组中的酵母和大肠杆菌细胞开发的基于cre/lox的新型遗传分析的效率和高灵敏度，作为与其他三个RBL小组的团队努力，以及对RNAP突变体、转录因子和促进这些生物中转录错误的反应条件的生化分析的能力和精确度。我们还在体外研究了高等真核生物转录保真度的机制。对于遗传分析，我们使用先进的高精度基因操作方法创建定点染色体突变和基因结构，该方法基于与Don Court的团队合作开发的重组工程。现代NGS RNA测序技术的应用将我们对转录错误的分析带到了基因组范围内。利用四个生物学模型，即大肠杆菌、枯草杆菌、酿酒酵母和人类三重阴性乳腺癌(TNBC)细胞在正常和应激条件下，允许我们对转录保真度进行跨物种和跨王国的研究，有助于揭示各种生物实施的控制转录保真度的保守和独特的策略。我们相信，我们在当前工作中使用的实验策略和技术为我们未来对转录保真度的研究奠定了坚实的基础。转录暂停：转录可以通过暂停RNAP而暂时停止，这为基因调控事件的发生提供了额外的时间。本项目涉及系统分析一般转录因子NusA、NusG、NusB、SuhB和Rho在启动后水平对转录的调控作用。在本研究中，我们以大肠杆菌和枯草杆菌细胞作为模型系统。今年，我们完成并发表了一篇论文，描述了枯草杆菌NusG在转录控制中的重要作用。NusG是一种普遍保守的转录延伸因子，在枯草芽孢杆菌基因组的两个位置刺激暂停，这两个位置都调节下游基因的表达。利用新生RNA的全基因组测序，我们在枯草杆菌中鉴定了数千个暂停位点，其中包括1600个依赖于NusG的暂停位点。NusG诱导RNAP暂停，以响应暂停转录泡中非模板DNA链中保守的TTNTTT序列基序。NusG依赖的停顿在体外的几个停顿位置被证实。核糖开关中NusG依赖的停顿降低了调节核糖表达所需的黄素单核苷酸浓度。

Transcription fidelity: In our approach to study transcription fidelity, we combine the efficiency and high-sensitivity of the novel cre/lox-based genetic assay, developed for the yeast and E. coli cells in our group, as a team effort with three other RBL groups, with the power and precision of biochemical analysis of RNAP mutants, transcription factors and reaction conditions promoting transcription errors in these organisms. We also study in vitro the mechanisms of transcription fidelity in higher eukaryotes. For genetic assay, we create site-directed chromosomal mutants and gene constructs using advanced high-precision methods of gene manipulations based on recombineering developed in collaboration with Don Court's group. Application of the modern NGS RNA sequencing techniques brings our analysis of transcription errors to a genome-wide scale. The use of four biological models, namely E. coli, B. subtilis, S. cerevisiae and human triple negative breast cancer (TNBC) cells under normal and stress conditions, allows us to do the cross-species and cross-kingdom study of transcription fidelity helping to unravel conserved and unique features of strategies that various organisms implement to control transcription fidelity. We believe that the experimental strategy and techniques that we use in the current work lay a solid foundation for our future studies on transcription fidelity. Transcription pausing: Transcription can be transiently halted by pausing of RNAP, which provides additional time for gene regulatory events to occur. This project involves a systematic analysis of the role of general transcription factors NusA, NusG, NusB, SuhB and Rho in control of transcription at post-initiation level. We use E. coli and B. subtilis cells as model systems in this study. This year we completed and published paper describing an important role of B. subtilis NusG in control of transcription. NusG is a universally conserved transcription elongation factor that was known to stimulate pausing at two positions in the Bacillus subtilis genome, both of which regulated expression of the downstream gene. Using genome-wide sequencing of nascent RNA, we identified thousands of pause sites in B. subtilis including 1,600 NusG-dependent pause sites. NusG induces pausing of RNAP in response to a conserved TTNTTT sequence motif in the non-template DNA strand within the paused transcription bubble. NusG-dependent pausing was confirmed at several pause sites in vitro. NusG-dependent pausing in the ribD riboswitch decreases the concentration of flavin mono-nucleotide required to regulate ribD expression.