Abstract
The transcriptome-wide contributions of Rho-dependent and intrinsic (Rho-independent) transcription termination mechanisms in bacteria are unclear. By sequencing released transcripts in a wild-type strain and strains containing deficiencies in NusA, NusG and/or Rho (10 strains), we produced an atlas of terminators for the model Gram-positive bacterium Bacillus subtilis. We found that NusA and NusG stimulate 77% and 19% of all intrinsic terminators, respectively, and that both proteins participate in Rho-dependent termination. We also show that Rho stimulates termination at 10% of the intrinsic terminators in vivo. We recapitulated Rho-stimulated intrinsic termination at 5 terminators in vitro and found that Rho requires the KOW domain of NusG to stimulate this process at one of these terminators. Computational analyses of our atlas using RNAstructure, MEME suite and DiffLogo, combined with in vitro transcription experiments, revealed that Rho stimulates intrinsic terminators with weak hairpins and/or U-rich tracts by remodelling the RNA upstream of the intrinsic terminator to prevent the formation of RNA structures that could otherwise compete with the terminator hairpin. We also identified 56 putative examples of ‘hybrid Rho-dependent termination’, wherein classical Rho-dependent termination occurs after readthrough of a Rho-stimulated intrinsic terminator.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 digital issues and online access to articles
$119.00 per year
only $9.92 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Data availability
Sequencing libraries generated in this study are deposited in Gene Expression Omnibus (GEO) (accession entry GSE188366). Previously published datasets for nusAdep and ΔnusG strains can be found at GEO with accession code GSE154522. Source data are provided with this paper.
Code availability
All scripts used to identify total 3’ ends from Term-seq data and calculate their termination efficiencies are archived on GitHub (https://github.com/zfmandell/Term-seq/releases/tag/v1.0).
References
Ray-Soni, A., Bellecourt, M. J. & Landick, R. Mechanisms of bacterial transcription termination: all good things must end. Annu. Rev. Biochem. 85, 319–347 (2016).
Skordalakes, E. & Berger, J. M. Structure of the Rho transcription terminator. Cell 114, 135–146 (2003).
Chen, C. Y. & Richardson, J. P. Sequence elements essential for ρ-dependent transcription termination at λtR1. J. Biol. Chem. 262, 11292–11299 (1987).
Das, A., Merril, C. & Adhya, S. Interaction of RNA polymerase and rho in transcription termination: coupled ATPase. Proc. Natl Acad. Sci. USA 75, 4828–4832 (1978).
Said, N. et al. Steps toward translocation-independent RNA polymerase inactivation by terminator ATPase ρ. Science 371, eabd1673 (2021).
Hao et al. Pre-termination transcription complex: structure and function. Mol. Cell 81, 281–292.e8 (2021).
Gusarov, I. & Nudler, E. The mechanism of intrinsic transcription termination. Mol. Cell 3, 495–504 (1999).
Jin, D. J., Burgess, R. R., Richardson, J. P. & Gross, C. A. Termination efficiency at rho-dependent terminators depends on kinetic coupling between RNA polymerase and rho. Proc. Natl Acad. Sci. USA 89, 1453–1457 (1992).
Fisher, R. F. & Yanofsky, C. Mutations of the β subunit of RNA polymerase alter both transcription pausing and transcription termination in the trp operon leader region in vitro. J. Biol. Chem. 258, 8146–8150 (1983).
Landick, R. & Yanofsky, C. Stability of an RNA secondary structure affects in vitro transcription pausing in the trp operon leader region. J. Biol. Chem. 259, 11550–11555 (1984).
Yakhnin, A. V. et al. NusG controls transcription pausing and RNA polymerase translocation throughout the Bacillus subtilis genome. Proc. Natl Acad. Sci. USA 117, 21628–21636 (2020).
Yakhnin, A. V. & Babitzke, P. NusA-stimulated RNA polymerase pausing and termination participates in the Bacillus subtilis trp operon attenuation mechanism in vitro. Proc. Natl Acad. Sci. USA 99, 11067–11072 (2002).
Saba et al. The elemental mechanism of transcriptional pausing. eLife 8, e40981 (2019).
Mondal, S., Yakhnin, A. V., Sebastian, A., Albert, I. & Babitzke, P. NusA-dependent transcription termination prevents misregulation of global gene expression. Nat. Microbiol. 1, 15007 (2016).
Mandell, Z. F. et al. NusG is an intrinsic transcription termination factor that stimulates motility and coordinates gene expression with NusA. eLife 10, e61880 (2021).
Guo, X. et al. Structural basis for NusA stabilized transcriptional pausing. Mol. Cell 69, 816–827 (2018).
Yakhnin, A. V., Murakami, K. S. & Babitzke, P. NusG is a sequence-specific RNA polymerase pause factor that binds to the non-template DNA within the paused transcription bubble. J. Biol. Chem. 291, 5299–5308 (2016).
Burmann, B. M. et al. A NusE:NusG complex links transcription and translation. Science 328, 501–504 (2010).
Sullivan, S. L. & Gottesman, M. E. Requirement for E. coli NusG protein in factor-dependent transcription termination. Cell 68, 989–994 (1992).
Arndt, K. M. & Chamberlin, M. J. RNA chain elongation by Escherichia coli RNA polymerase. Factors affecting the stability of elongating ternary complexes. J. Mol. Biol. 213, 79–108 (1990).
Komissarova, N., Becker, J., Solter, S., Kireeva, M. & Kashlev, M. Shortening of RNA:DNA hybrid in the elongation complex of RNA polymerase is a prerequisite for transcription termination. Mol. Cell 10, 1151–1162 (2002).
Santangelo, T. J. & Roberts, J. W. Forward translocation is the natural pathway of RNA release at an intrinsic terminator. Mol. Cell 14, 117–126 (2004).
Park, J. S. & Roberts, J. W. Role of DNA bubble rewinding in enzymatic transcription termination. Proc. Natl Acad. Sci. USA 103, 4870–4875 (2006).
Richardson, J. P. Rho-dependent termination and ATPases in transcript termination. Biochim. Biophys. Acta 1577, 251–260 (2002).
Roberts, J. W. Mechanisms of bacterial transcription termination. J. Mol. Biol. 431, 4030–4039 (2019).
Peters, J. M., Vangeloff, A. D. & Landick, R. Bacterial transcription terminators: the RNA 3’-end chronicles. J. Mol. Biol. 412, 793–813 (2011).
Lodish, H. F. et al. Molecular Cell Biology 4th edn (W. H. Freeman, 2000).
Farnham, P. J., Greenblatt, J. & Platt, T. Effects of NusA protein on transcription termination in the tryptophan operon of Escherichia coli. Cell 29, 945–951 (1982).
Lau, L. F., Roberts, J. W. & Wu, R. Transcription terminates at λtR1 in three clusters. Proc. Natl Acad. Sci. USA 79, 6171–6175 (1982).
Wilson, K. S. & von Hippel, P. H. Transcription termination at intrinsic terminators: the role of the RNA hairpin. Proc. Natl Acad. Sci. USA 92, 8793–8797 (1995).
Fuller, R. S. & Platt, T. The attenuator of the tryptophan operon in E.coli: rho-mediated release of RNA polymerase from a transcription termination complex in vitro. Nucleic Acids Res. 5, 4613–4623 (1978).
Korn, L. J. & Yanofsky, C. Polarity suppressors defective in transcription termination at the attenuator of the tryptophan operon of Escherichia coli have altered Rho factor. J. Mol. Biol. 106, 231–241 (1976).
Ju, X., Li, D. & Liu, S. Full-length RNA profiling reveals pervasive bidirectional transcription terminators in bacteria. Nat. Microbiol. 4, 1907–1918 (2019).
Ingham, C. J., Dennis, J. & Furneaux, P. A. Autogenous regulation of transcription termination factor Rho and the requirement for Nus factors in Bacillus subtilis. Mol. Microbiol. 31, 651–663 (1999).
Dar, D. et al. Term-seq reveals abundant ribo-regulation of antibiotics resistance in bacteria. Science 352, aad9822 (2016).
Bidnenko, V. et al. Termination factor Rho: from the control of pervasive transcription to cell fate determination in Bacillus subtilis. PLoS Genet. 13, e1006909 (2017).
Nadiras, C., Eveno, E., Schwartz, A., Figueroa-Bossi, N. & Boudvillain, M. A multivariate prediction model for Rho-dependent termination of transcription. Nucleic Acids Res. 46, 8245–8260 (2018).
Liu, B., Kearns, D. B. & Bechhofer, D. H. Expression of multiple Bacillus subtilis genes is controlled by decay of slrA mRNA from Rho-dependent 3’ ends. Nucleic Acids Res. 44, 3364–3372 (2016).
Dar, D. & Sorek, R. High-resolution RNA 3′-ends mapping of bacterial Rho-dependent transcripts. Nucleic Acids Res. 46, 6797–6805 (2018).
Chhabra, S., Mandell, Z. F., Liu, B., Babitzke, P. & Bechhofer, D. H. Analysis of mRNA decay intermediates in Bacillus subtilis 3′ exoribonuclease and RNA helicase mutant strains. mBio 13, e0040022 (2022).
Ingle, S. et al. Polynucleotide phosphorylase and RNA helicase CshA cooperate in Bacillus subtilis mRNA decay. RNA Biol. 18, 1692–1701 (2021).
Wang, X. et al. Processing generates 3′ ends of RNA masking transcription termination events in prokaryotes. Proc. Natl Acad. Sci. USA 116, 4440–4445 (2019).
Toulokhonov, I., Artsimovitch, I. & Landick, R. Allosteric control of RNA polymerase by a site that contacts nascent RNA hairpins. Science 292, 730–733 (2001).
Reuter, J. S. & Mathews, D. H. RNAstructure: software for RNA secondary structure prediction and analysis. BMC Bioinformatics 11, 129 (2010).
Chen, Y. J. et al. Characterization of 582 natural and synthetic terminators and quantification of their design constraints. Nat. Methods 10, 659–664 (2013).
Bailey, T. L. & Elkan, C. Fitting a mixture model by expectation maximization to discover motifs in biopolymers. Proc. Int. Conf. Intell. Syst. Mol. Biol. 2, 28–36 (1994).
Nettling, M. et al. DiffLogo: a comparative visualization of sequence motifs. BMC Bioinformatics 16, 387 (2015).
Johnson, G. E., Lalanne, J., Peters, M. L. & Li, G. Functionally uncoupled transcription–translation in Bacillus subtilis. Nature 585, 124–128 (2020).
Zhu, M., Mu, H., Han, F., Wang, Q. & Xiongfeng, D. Quantitative analysis of asynchronous transcription-translation and transcription processivity in Bacillus subtilis under various growth conditions. iScience 24, 103333 (2021).
Santangelo, T. J. & Artsimovitch, I. Termination and antitermination: RNA polymerase runs a stop sign. Nat. Rev. Microbiol. 9, 319–329 (2011).
Winkler, W. C., Cohen-Chalamish, S. & Breaker, R. R. An mRNA structure that controls gene expression by binding FMN. Proc. Natl Acad. Sci. USA 99, 15908–15913 (2002).
Lalanne, J. B. et al. Evolutionary convergence of pathway-specific enzyme expression stoichiometry. Cell 173, 749–761 (2018).
Skordalakes, E., Brogan, A. P., Park, B. S., Kohn, H. & Berger, J. M. Structural mechanism of inhibition of the Rho transcription termination factor by the antibiotic bicyclomycin. Structure 13, 99–109 (2005).
Takyar, S., Hickerson, R. P. & Noller, H. F. mRNA helicase activity of the ribosome. Cell 120, 49–58 (2005).
Jayasinghe, O. T., Mandell, Z. F., Yakhnin, A. V., Kashlev, M. & Babitzke, P. Transcriptome-wide role of NusA-stimulated RNA polymerase pausing in Bacillus subtilis. J. Bacteriol. 204, e0053421 (2022).
Wu, A. M., Christie, G. E. & Platt, T. Tandem termination sites in the tryptophan operon of Escherichia coli. Proc. Natl Acad. Sci. USA 78, 2913–2917 (1981).
Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for illumina sequence data. Bioinformatics 30, 2114–2120 (2014).
Marcel, M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet 17, 10–12 (2011).
Li, H. Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM. Preprint at https://arxiv.org/abs/1303.3997v2 (2013).
Li, H. et al. The sequence alignment/map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).
Quinlan, A. R. & Hall, I. M. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010).
Bray, N. L., Pimentel, H., Melsted, P. & Pachter, L. Near-optimal probabilistic RNA-seq quantification. Nat. Biotechnol. 34, 525–527 (2016).
Ritchey, L. E. et al. Structure-seq2 probing of RNA structure upon amino acid starvation reveals both known and novel RNA switches in Bacillus subtilis. RNA 26, 1431–1447 (2020).
Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).
Lanzetta, P. A., Alvarez, L. J., Reinach, P. S. & Candia, O. A. An improved assay for nanomole amounts of inorganic phosphate. Anal. Biochem. 100, 95–97 (1979).
Lin, A. A. & Zuber, P. Evidence that a single monomer of Spx can productively interact with RNA polymerase in Bacillus subtilis. J. Bacteriol. 194, 1697–1707 (2012).
Acknowledgements
This work was supported by National Institutes of Health (NIH) Grant GM098399 (to P.B.), NIH grant GM131860 (to K.S.M.) and the Intramural Research Program of the NIH National Cancer Institute (to M.K.). We thank A. Yakhnin for critically reading the manuscript. Anti-σA antibodies and anti-NusA antibodies were obtained from M. Fujita (University of Houston) and P. Lewis (University of Newcastle), respectively. HRP-conjugated goat anti-rabbit antibody is available from GenScript. All bacterial strains are available from P.B.
Author information
Authors and Affiliations
Contributions
Z.F.M., M.K. and P.B. conceptualized the project. Z.F.M. and R.K.V. developed the methodology. Z.F.M, R.K.V., K.S.M., M.K. and P.B. conducted formal analyis. Z.F.M., R.K.V. and H.Y. conducted investigations. Z.F.M. wrote the original draft. Z.F.M, R.K.V., H.Y., K.S.M., M.K. and P.B. reviewed and edited the manuscript. K.S.M., M.K. and P.B. secured funding.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Peer review
Peer review information
Nature Microbiology thanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are available.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data
Extended Data Fig. 1 NusA depletion and benchmarking of terminators.
a. Western blot for all samples used for Term-seq was performed once. Top panel, image after probing for NusA. Bottom panel, image after probing for σA as a loading control. Lanes: 1, purified NusA6His (top panel) or σA (bottom panel); 2, PLBS730 + IPTG (WT); 3, PLBS730 –IPTG (nusAdep); 4, PLBS731 + IPTG (ΔnusG); 5, PLBS731 –IPTG (nusAdep ΔnusG); 6, PLBS890 + IPTG (Δrho); 7, PLBS890 –IPTG (nusAdep Δrho); 8, PLBS891 + IPTG (ΔnusG Δrho); 9, PLBS891 –IPTG (nusAdep ΔnusG Δrho). b. Venn diagram showing the number and overlap of intrinsic terminators identified in PLBS727 (WT in this study) and those identified previously in PLBS730 + IPTG (WT in previous study). c-f. RNA-seq coverage data from WT and Δrho strains across the previously identified classical Rho-dependent terminators for ylaL (c), spoVB (d), rapD (e) and slrA (f). Arrows at the bottom indicate the direction of transcription. Top tracks are the 3’ ends identified by Term-seq across each region.
Extended Data Fig. 2 A-rich tracts and Rho-stimulated intrinsic termination in vivo.
a. A-rich tract motifs generated for the FI, SA, SG, and SR intrinsic terminator subpopulations. A hierarchical clustering analysis can be found at the top of the motifs. b. IGV screenshot of the region upstream of the tbcS terminator. Top track is the 3’ end identified by Term-seq. Bottom tracks are the RNA-seq coverage data for the WT and Δrho strains. Arrow at the bottom indicates the direction of transcription. c. IGV screenshot of RNA-seq and ribosome profiling data across the transcript containing two sfp pseudogenes. Arrows at the bottom indicate the direction of transcription.
Extended Data Fig. 3 Verification of intrinsic terminator function in vitro.
a. Single-round in vitro termination assay with the tbcS intrinsic terminator. Experiments were performed with templates containing the WT terminator or those in which the DNA corresponding to the U-rich tract (ΔU) or the U-rich tract and the terminator hairpin (ΔU + HP) was deleted. Experiments were performed in the absence (–) or presence of Rho (R). b. Single-round in vitro termination assay with the yybG intrinsic terminator. Experiments were performed with WT or ΔU + HP templates in the absence (–) or presence of Rho (R), NusA (A) and/or NusG (G). c. Single-round in vitro termination assay with the bstB intrinsic terminator. Experiments were performed with WT, ΔHP or ΔU + HP templates in the absence (–) or presence of Rho (R). These qualitative intrinsic terminator validation experiments were performed once.
Extended Data Fig. 4 Rho-stimulated intrinsic terminators with antiterminators.
a. IGV screenshot of a genomic window centered around the yybG intrinsic terminator. Top track is the 3’ end identified by Term-seq. Bottom tracks are the RNA-seq coverage data for the WT and Δrho strains. %T in each strain is shown on the right of each track. Arrow at the bottom depicts the direction of transcription. b. Model of the yybG intrinsic terminator. c. Single-round in vitro termination assay with the yybG intrinsic terminator. Experiments were performed in the absence (–) or presence of Rho (R). Positions of terminated (Term) and full-length (FL) transcripts are marked. %T ± standard deviation is shown below each lane. Loss of termination in vitro when the terminator was deleted established that this is an authentic intrinsic terminator (Extended Data Fig. 3b). d-f. same as for panels a-c, except that it is the bstB (yuaE) intrinsic terminator. Loss of termination in vitro when the terminator was deleted established that this is an authentic intrinsic terminator (Extended Data Fig. 3c). g. RNA sequence of the ribD, sfp, tbcS, yybG and bstB leader terminators (inverted arrows). Red sequences can participate in the formation of alternative antiterminator (AT) structures. In vitro transcription experiments were performed 3 times. Values are averages ± standard deviation.
Extended Data Fig. 5 Sequencing data from each strain and Rho ATPase assay.
a. Principal component analysis (PCA) plot of transcriptomics data collected from each Term-seq replicate. b. Bar graph showing the nmol of Pi that was released by Rho in the presence of a polyC transcript and in the absence (–) or presence of BCM. ATPase assays were performed 3 times. Values are averages ± standard deviation.
Supplementary information
Supplementary Table 1
All classical Rho-dependent terminators identified in the wild-type strain.
Supplementary Table 2
All intrinsic terminators identified in the wild-type strain.
Supplementary Table 3
Bacterial strains, oligonucleotides, plasmids and P values.
Supplementary Table 4
Analysis of rut sites at Rho-stimulated intrinsic terminators and all intrinsic terminators identified in the wild-type strain and expression data for all strains.
Supplementary Table 5
Results of differential expression analysis for all mutant strains compared to the wild-type strain.
Source data
Source Data Fig. 1
Unprocessed gels for Fig. 4.
Source Data Fig. 2
Unprocessed gels for Fig. 5.
Source Data Extended Data Fig. 1
Unprocessed western Blots for Extended Data Fig. 1.
Source Data Extended Data Fig. 2
Unprocessed gels for Extended Data Fig. 3.
Source Data Extended Data Fig. 3
Unprocessed gels for Extended Data Fig. 4.
Rights and permissions
Springer Nature or its licensor holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Mandell, Z.F., Vishwakarma, R.K., Yakhnin, H. et al. Comprehensive transcription terminator atlas for Bacillus subtilis. Nat Microbiol 7, 1918–1931 (2022). https://doi.org/10.1038/s41564-022-01240-7
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41564-022-01240-7
This article is cited by
-
Co-transcriptional gene regulation in eukaryotes and prokaryotes
Nature Reviews Molecular Cell Biology (2024)
-
Extensive diversity in RNA termination and regulation revealed by transcriptome mapping for the Lyme pathogen Borrelia burgdorferi
Nature Communications (2023)