期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2021
卷号：118
期号：45
DOI：10.1073/pnas.2106564118
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Significance
Transcription of DNA into RNA is crucial to life, and understanding RNA polymerase (RNAP) function has received considerable attention. In contrast, how the nascent RNA folds into structures that impact transcription itself and regulate gene expression remains poorly understood. Here, we combine single-molecule Förster resonance energy transfer and site-specific fluorescent labelling of transcripts within native complexes to enable real-time cotranscriptional folding studies of a metabolite-sensing riboswitch from
Escherichia coli. By monitoring the folding of riboswitches stalled at RNAP pausing sites and during active elongation, we reveal a crucial role for RNAP, which directs RNA folding to allow thiamin pyrophosphate sensing within a precise, transcriptional hotspot. Our approach offers a unique opportunity to unveil cotranscriptional processes in eukaryotic and bacterial systems.
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
Escherichia coli 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.
