Investigating the role of RNA structures in transcriptional pausing using in vitro assays and in silico analyses

Transcriptional pausing occurs across the bacterial genome but the importance of this mechanism is still poorly understood. Only few pauses were observed during the previous decades, leaving an important gap in understanding transcription mechanisms. Using the well-known <i>Escherichia coli hisL</i> and <i>trpL</i> pause sites as models, we describe here the relation of pause sites with upstream RNA structures suspected to stabilize pausing. We find that the transcription factor NusA influences the pause half-life at <i>leuL, pheL</i> and <i>thrL</i> pause sites. Using a mutagenesis approach, we observe that transcriptional pausing is affected in all tested pause sites, suggesting that the upstream RNA sequence is important for transcriptional pausing. Compensatory mutations assessing the presence of RNA hairpins did not yield clear conclusions, indicating that complex RNA structures or transcriptional features may be playing a role in pausing. Moreover, using a bioinformatic approach, we explored the relation between a DNA consensus sequence important for pausing and putative hairpins among thousands of pause sites in <i>E. coli</i>. We identified 2125 sites presenting hairpin-dependent transcriptional pausing without consensus sequence, suggesting that this mechanism is widespread across <i>E. coli</i>. This study paves the way to understand the role of RNA structures in transcriptional pausing.

转录暂停现象广泛分布于细菌基因组中，但该机制的生物学重要性目前仍未被充分认知。过往数十年间，仅观测到少量转录暂停事件，这为完整解析转录机制留下了重要的研究空白。本研究以经典的大肠杆菌（Escherichia coli）hisL与trpL暂停位点作为模型，探讨了暂停位点与疑似可稳定转录暂停过程的上游RNA二级结构之间的关联。我们发现，转录因子NusA可调控leuL、pheL及thrL暂停位点处的暂停半衰期。通过诱变实验方法，我们观测到所有受试暂停位点的转录暂停过程均受到影响，这表明上游RNA序列对转录暂停过程具有关键作用。针对RNA发夹结构（RNA hairpins）存在性的补偿突变实验未得到明确结论，这提示复杂RNA结构或其他转录相关特征可能参与了转录暂停的调控。此外，本研究借助生物信息学方法，在大肠杆菌的数千个暂停位点中，探究了与转录暂停相关的DNA共有序列与推定发夹结构之间的关联。我们共鉴定出2125个存在发夹依赖型转录暂停但无共有序列的位点，这表明该机制在大肠杆菌中广泛存在。本研究为理解RNA结构在转录暂停过程中的作用铺平了道路。