Department of Biology, Faculty of Science, RNA Group, Université de Sherbrooke, Sherbrooke, QC J1K 2R1, Canada.
Centre of Biophotonics, Laboratory for Biophysics and Biomolecular Dynamics, Scottish Universities Physics Alliance (SUPA) School of Physics and Astronomy, University of St. Andrews, St. Andrews KY16 9SS, United Kingdom.
Proc Natl Acad Sci U S A. 2021 Nov 9;118(45). doi: 10.1073/pnas.2106564118.
Cotranscriptional RNA folding is crucial for the timely control of biological processes, but because of its transient nature, its study has remained challenging. While single-molecule Förster resonance energy transfer (smFRET) is unique to investigate transient RNA structures, its application to cotranscriptional studies has been limited to nonnative systems lacking RNA polymerase (RNAP)-dependent features, which are crucial for gene regulation. Here, we present an approach that enables site-specific labeling and smFRET studies of kilobase-length transcripts within native bacterial complexes. By monitoring nascent riboswitches, we reveal an inverse relationship between elongation speed and metabolite-sensing efficiency and show that pause sites upstream of the translation start codon delimit a sequence hotspot for metabolite sensing during transcription. Furthermore, we demonstrate a crucial role of the bacterial RNAP actively delaying the formation, within the hotspot sequence, of competing structures precluding metabolite binding. Our approach allows the investigation of cotranscriptional regulatory mechanisms in bacterial and eukaryotic elongation complexes.
共转录 RNA 折叠对于及时控制生物过程至关重要，但由于其瞬态性质，其研究一直具有挑战性。虽然单分子Förster 共振能量转移（smFRET）是研究瞬时 RNA 结构的独特方法，但将其应用于共转录研究仅限于缺乏 RNA 聚合酶（RNAP）依赖性特征的非天然系统，这些特征对于基因调控至关重要。在这里，我们提出了一种方法，可实现天然细菌复合物中千碱基长度转录本的定点标记和 smFRET 研究。通过监测新生的核酶开关，我们揭示了延伸速度和代谢物感应效率之间的反比关系，并表明翻译起始密码子上游的暂停位点限定了转录过程中代谢物感应的序列热点。此外，我们证明了细菌 RNAP 主动延迟形成竞争结构的关键作用，这些结构阻止了代谢物结合。我们的方法允许在细菌和真核延伸复合物中研究共转录调控机制。
