The ability to determine full-length nucleotide composition of individual RNA molecules is essential for understanding the architecture and function of a transcriptome. However, experimental approaches capable of capturing the sequences of both 5′ and 3′ termini of the same transcript remain scarce. In the present study, simultaneous 5′ and 3′ end sequencing (SEnd-seq)—a high-throughput and unbiased method that simultaneously maps transcription start and termination sites with single-nucleotide resolution—is presented. Using this method, a comprehensive view of the Escherichia coli transcriptome was obtained, which displays an unexpected level of complexity. SEnd-seq notably expands the catalogue of transcription start sites and termination sites, defines unique transcription units and detects prevalent antisense RNA. Strikingly, the results of the present study unveil widespread overlapping bidirectional terminators located between opposing gene pairs. Furthermore, it has been shown that convergent transcription is a major contributor to highly efficient bidirectional termination both in vitro and in vivo. This finding highlights an underappreciated role of RNA polymerase conflicts in shaping transcript boundaries and suggests an evolutionary strategy for modulating transcriptional output by arranging gene orientation.
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SEnd-seq and standard RNA-seq datasets from this study have been deposited in the Gene Expression Omnibus (GEO) with the accession number GSE117737.
The custom scripts used in this study are available on Github (https://github.com/LiuLab-codes/SEnd_seq_analysis). Other data that support the findings of this study are available from the corresponding author upon request.
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We thank S. Darst and E. Campbell for help with the in vitro transcription experiments and critical reading of the manuscript, K. Ryan, A. Pyle and R. Landick for sharing reagents and E. Cheng for help with data analysis. This work was supported by a C.H. Li Memorial Scholar Fund Award (X.J.), the Robertson Foundation, the Quadrivium Foundation, a Monique Weill-Caulier Career Scientist Award, a March of Dimes Basil O’Connor Starter Scholar Award, a Kimmel Scholar Award, and National Institute of Health grants R00GM107365 and DP2HG010510 (S.L.).
The Rockefeller University has filed a provisional patent application encompassing aspects of the SEnd-seq technology on which S.L. and X.J. are listed as inventors.
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Supplementary Figs. 1–23, Supplementary Tables 1–5 and Supplementary References.
Genomic position of TSSs identified by SEnd-seq.
Genomic position and predicted secondary structure of TTSs identified by SEnd-seq.
Transcription units defined by SEnd-seq.
Genomic position and structural analysis of overlapping bidirectional TTSs and the E. coli genes involved.
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Ju, X., Li, D. & Liu, S. Full-length RNA profiling reveals pervasive bidirectional transcription terminators in bacteria. Nat Microbiol 4, 1907–1918 (2019). https://doi.org/10.1038/s41564-019-0500-z
Nature Reviews Genetics (2021)
Nature Reviews Genetics (2020)