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NusA-dependent transcription termination prevents misregulation of global gene expression

Nature Microbiology volume 1, Article number: 15007 (2016) Cite this article

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A Corrigendum to this article was published on 13 June 2016

Abstract

Intrinsic transcription terminators consist of an RNA hairpin followed by a U-rich tract, and these signals can trigger termination without the involvement of additional factors. Although NusA is known to stimulate intrinsic termination in vitro, the in vivo targets and global impact of NusA are not known because it is essential for viability. Using genome-wide 3′ end-mapping on an engineered Bacillus subtilis NusA depletion strain, we show that weak suboptimal terminators are the principle NusA substrates. Moreover, a subclass of weak non-canonical terminators was identified that completely depend on NusA for effective termination. NusA-dependent terminators tend to have weak hairpins and/or distal U-tract interruptions, supporting a model in which NusA is directly involved in the termination mechanism. Depletion of NusA altered global gene expression directly and indirectly via readthrough of suboptimal terminators. Readthrough of NusA-dependent terminators caused misregulation of genes involved in essential cellular functions, especially DNA replication and metabolism. We further show that nusA is autoregulated by a transcription attenuation mechanism that does not rely on antiterminator structures. Instead, NusA-stimulated termination in its 5′ UTR dictates the extent of transcription into the operon, thereby ensuring tight control of cellular NusA levels.

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Figure 1: Depletion of NusA and its effect at intrinsic terminators in vivo.
Figure 2: Effect of NusA on intrinsic termination in vivo and in vitro.
Figure 3: Terminator features contributing to stimulation by NusA.
Figure 4: NusA depletion alters expression of genes downstream of intrinsic terminators.
Figure 5: Autoregulation of nusA expression by NusA-mediated transcription attenuation.

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Acknowledgements

Illumina library preparation and sequencing was performed at the Penn State Genomics Core Facility. The authors thank P. Lewis and M. Fujita for providing NusA and σA antibodies, respectively. E. coli NusA was provided by C. Squires. This work was supported by National Institutes of Health grant GM098399 to P.B.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, 16802, Pennsylvania, USA

    Smarajit Mondal, Alexander V. Yakhnin, Istvan Albert & Paul Babitzke

  2. Center for RNA Molecular Biology, The Pennsylvania State University, University Park, 16802, Pennsylvania, USA

    Smarajit Mondal, Alexander V. Yakhnin & Paul Babitzke

  3. Bioinformatics Consulting Center, The Pennsylvania State University, University Park, 16802, Pennsylvania, USA

    Aswathy Sebastian & Istvan Albert

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  1. Smarajit Mondal
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Contributions

S.M. performed all experiments except for illumina library generation and sequencing. A.V.Y. discovered NusA-dependent termination in vitro. A.S. and I.A. processed raw sequencing data, mapped the sequencing reads to the genome and developed computational algorithms. P.B. and S.M. analysed the processed data. S.M. and P.B. wrote the manuscript. P.B. supervised the project.

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Correspondence to Paul Babitzke.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Methods, References and Figures 1–6. (PDF 1568 kb)

Supplementary Table 1

RNA 3′ ends identified by 3′ end-mapping. (XLSX 241 kb)

Supplementary Table 2

The effect of NusA on terminators. (XLSX 289 kb)

Supplementary Table 3

Genes that are differentially expressed twofold or more (p-adj < 0.05) upon NusA depletion. (XLSX 95 kb)

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Mondal, S., Yakhnin, A., Sebastian, A. et al. NusA-dependent transcription termination prevents misregulation of global gene expression. Nat Microbiol 1, 15007 (2016). https://doi.org/10.1038/nmicrobiol.2015.7

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