Importance of transcriptional pausing in bacterial riboswitch regulation

核糖开关是高度结构化的基序，通常存在于信使RNA的非编码区，在那里它们结合代谢物并控制基因表达。与大多数其他遗传控制系统不同，核糖开关不直接需要蛋白质作为细胞传感器，因此在信使RNA编码的遗传信息与其细胞环境之间提供了直接联系。已经发现核糖开关的活性依赖于转录过程，转录过程取决于几个参数，例如RNA聚合酶（RNAP）、延伸速度和转录暂停。这项研究的目的首先是利用大肠杆菌核糖开关来表征几种大肠杆菌核糖开关中的转录暂停。coliRNAP以及代谢物结合对停顿的影响。特别地，将使用高转录速度作为接近接近生理条件的方式来表征转录暂停。将在有和没有核糖开关同源配体的情况下测量核糖开关转录暂停位点的半衰期，以确定这些暂停位点如何受到配体结合的影响。第二个目标将是确定转录因子，如NusA，可能参与核糖开关转录暂停。拟议的研究的第三个目标将是实现使用光激活的NPOM笼状DNA的转录暂停位点的表征。在转录研究中使用NPOM将允许直接测量暂停位点的半衰期，而不会因异步转录反应的存在而引起并发症。我们期望通过监测不同条件下的转录暂停来获得有关核糖开关调控过程的重要信息。我们的工作将提供新的见解RNA折叠和代谢物传感机制，以及它是如何参与核糖开关依赖的控制基因表达。

Riboswitches are highly structured motifs that are usually found in the non-coding region of messenger RNA, where they bind metabolites and control gene expression. Unlike most other genetic control systems, riboswitches do not directly require proteins to act as cellular sensors, and thus provide a direct link between the genetic information that is encoded by a messenger RNA and its cellular environment. The activity of riboswitches has been found to rely on the transcriptional process, which depends on several parameters such as the RNA polymerase (RNAP), elongation speed and transcriptional pausing. The proposed research will first aim to characterize transcriptional pausing within several Escherichia coli riboswitches using the E. coli RNAP and the influence of metabolite binding on pausing. In particular, transcriptional pausing will be characterized using a high transcriptional speed as a way to approach near-physiological conditions. The half-life of riboswitch transcriptional pause sites will be measured with and without riboswitch cognate ligands, to establish how such pause sites are influenced by ligand binding. The second goal will be to determine how transcription factors, such as NusA, might be involved in riboswitch transcriptional pausing. The third goal of the proposed research will be to implement the use of light-activated NPOM-caged DNA for the characterization of transcriptional pause sites. The use of NPOM in transcriptional studies would allow to directly measure the half-life of pause sites without complications that could arise by the presence of asynchronous transcription reactions. We expect to obtain vital information about riboswitch regulatory processes by monitoring transcriptional pausing under different conditions. Our work will provide new insights on RNA folding and metabolite sensing mechanisms, and how it is involved in the riboswitch-dependent control of genetic expression.