Importance of transcriptional pausing in bacterial riboswitch regulation

点击翻译按钮获取中文摘要

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.