REFERENCES AND NOTES
1. K. H. Nealson, A. Belz, B. McKee, Antonie van Leeuwenhoek 81,
2. D. R. Lovley, in The Prokaryotes, E. Rosenberg, E. F. DeLong,
S. Lory, E. Stackebrandt, F. Thompson, Eds. (Springer,
Berlin, 2013), pp. 287–308.
3. M. F. Kirk, L. J. Crossey, C. Takacs-Vesbach, D. L. Newell,
R. S. Bowman, Appl. Geochem. 24, 426–437 (2009).
4. T. Borch et al., Environ. Sci. Technol. 44, 15–23 (2010).
5. K. J. Edwards, K. Becker, F. Colwell, Annu. Rev. Earth Planet.
Sci. 40, 551–568 (2012).
6. A. Heimann et al., Earth Planet. Sci. Lett. 294, 8–18 (2010).
7. K. H. Nealson, Annu. Rev. Earth Planet. Sci. 25, 403–434
8. K. L. Straub, B. Schink, Appl. Environ. Microbiol. 70, 5744–5749
9. S. A. Haveman et al., Appl. Environ. Microbiol. 74, 4277–4284
10. S. W. Poulton, M. D. Krom, R. Raiswell, Geochim. Cosmochim.
Acta 68, 3703–3715 (2004).
11. J. L. Burns, T. J. DiChristina, Appl. Environ. Microbiol. 75,
12. Q. Jin, C. M. Bethke, Am. J. Sci. 307, 643–677 (2007).
13. T. M. Hoehler, B. B. Jørgensen, Nat. Rev. Microbiol. 11, 83–94
14. C. M. Bethke, R. A. Sanford, M. F. Kirk, Q. Jin, T. M. Flynn,
Am. J. Sci. 311, 183–210 (2011).
15. J. N. Andrews et al., Water Resour. Res. 30, 45–61 (1994).
16. A. H. Welch, D. B. Westjohn, D. R. Helsel, R. B. Wanty, Ground
Water 38, 589–604 (2000).
17. M. F. Kirk et al., Geology 32, 953 (2004).
18. B. P. McGrail et al., J. Geophys. Res. Solid Earth 111, B12201
19. A. J. Williamson et al., Appl. Environ. Microbiol. 79, 3320–3326
20. S. J. Fuller et al., Appl. Environ. Microbiol. 80, 128–137 (2014).
21. P. B. McMahon, F. H. Chapelle, Nature 349, 233–235 (1991).
22. S. W. Poulton, Chem. Geol. 202, 79–94 (2003).
23. K. Hellige, K. Pollok, P. Larese-Casanova, T. Behrends,
S. Peiffer, Geochim. Cosmochim. Acta 81, 69–81 (2012).
24. M. Ledin, K. Pedersen, Earth Sci. Rev. 41, 67–108 (1996).
25. R. Jakobsen, D. Postma, Geochim. Cosmochim. Acta 63,
26. T. M. Flynn et al., BMC Microbiol. 13, 146 (2013).
27. S. Kempe, E. T. Degens, Chem. Geol. 53, 95–108 (1985).
28. C. M. Johnson, B. L. Beard, E. E. Roden, Annu. Rev. Earth
Planet. Sci. 36, 457–493 (2008).
29. L. Wu, B. L. Beard, E. E. Roden, C. M. Johnson, Geochim.
Cosmochim. Acta 73, 5584–5599 (2009).
30. M. J. Bickle, Nat. Geosci. 2, 815–818 (2009).
This research is part of the Subsurface Science Scientific Focus
Area at Argonne National Laboratory supported by the Subsurface
Biogeochemical Research Program, U.S. Department of Energy
(DOE) Office of Science, Office of Biological and Environmental
Research, under DOE contract DE-AC02-06CH11357. We
appreciate the technical assistance of M. Newville, and
A. Lanzirotti. K. Nealson, J. Fredrickson, and K. Haugen provided
helpful comments that improved the manuscript. X-ray analyses
were conducted at Argonne National Laboratory’s Advanced
Photon Source (APS), GeoSoilEnviroCARS (Sector 13), supported
by NSF–Earth Sciences (EAR-1128799) and DOE–GeoSciences
(DE-FG02-94ER14466). Use of the APS was supported by the DOE
Office of Science, Office of Basic Energy Sciences. T.F. was
supported in part by an Argonne Director’s Fellowship and the
National Institute of Allergy and Infectious Diseases, NIH,
Department of Health and Human Service (contract no.
HHSN272200900040C). T.D. was supported by NSF (Molecular
and Cellular Biosciences grant no. 1021735). All additional data
have been archived in the supplementary materials.
Materials and Methods
Figs. S1 and S2
Tables S1 and S2
11 February 2014; accepted 18 April 2014
Published online 1 May 2014;
A pause sequence enriched at
translation start sites drives
transcription dynamics in vivo
Matthew H. Larson,1 Rachel A. Mooney,2 Jason M. Peters,3 Tricia Windgassen,2
Dhananjaya Nayak,2 Carol A. Gross,3 Steven M. Block,4,5 William J. Greenleaf,6*
Robert Landick,2,7 Jonathan S. Weissman1*
Transcription by RNA polymerase (RNAP) is interrupted by pauses that play diverse
regulatory roles. Although individual pauses have been studied in vitro, the determinants
of pauses in vivo and their distribution throughout the bacterial genome remain unknown.
Using nascent transcript sequencing, we identified a 16-nucleotide consensus pause
sequence in Escherichia coli that accounts for known regulatory pause sites as well as
~20,000 new in vivo pause sites. In vitro single-molecule and ensemble analyses
demonstrate that these pauses result from RNAP–nucleic acid interactions that inhibit
next-nucleotide addition. The consensus sequence also leads to pausing by RNAPs from
diverse lineages and is enriched at translation start sites in both E. coli and Bacillus
subtilis. Our results thus reveal a conserved mechanism unifying known and newly
identified pause events.
Transcriptional pausing by RNA polymerase (RNAP) is an important feature of gene regulation that facilitates RNA folding (1), factor recruitment (2), transcription termination (3), and synchronization with
translation in prokaryotes (4, 5). Previously characterized regulatory pauses (6) represent a very
small and biased fraction of potential pause
sites in the bacterial genome. Furthermore, it
remains unknown whether most pauses identified by in vitro studies affect transcription in vivo.
To study transcriptional pausing in vivo, we
adapted a high-throughput approach to isolate
and sequence nascent elongating transcripts
(NET-seq) (7). Escherichia coli nascent transcripts
were captured by immunoprecipitating FLAG-tagged RNAP molecules, converted to DNA, and
sequenced to a depth of ~30 million reads per
sample (figs. S1 to S3 and tables S1 and S2). Each
sequencing read was mapped to a single site corresponding to the 3′ end of the nascent transcript
(Fig. 1A), allowing us to define RNAP locations
along ~2000 genes with single-nucleotide resolution (table S2).
The number of mapped reads at each genomic
position is proportional to the number of RNAP
molecules at that position. We observed well-defined single-nucleotide peaks within transcribed
regions at known regulatory pause sites, including sites that synchronize transcription with
translation, mediate RNA folding, or recruit transcription factors (Fig. 1B and fig. S4, A to E). NET-seq profiles also revealed a large number of
other highly reproducible peaks in RNAP density
throughout the genome (example gene in Fig. 1C).
In total, we identified ~20,000 previously undocumented pause sites across well-transcribed genes,
representing an average frequency of 1 per 100
base pairs (bp) (Fig. 1D). Thus, known regulatory pause sites represent a tiny fraction of actual
We found that in vivo pause propensity depended strongly on the sequence identity at the
3′ end of the transcript (87% of paused transcripts
end with either cytosine or uracil), as well as on
the identity of the incoming nucleoside triphosphate (NTP) substrate [70% of pause sites occur before addition of guanosine 5′-triphosphate
(GTP)] (Fig. 2A). Sequence dependence extends
outside the RNAP active site to 11 nucleotides
(nt) upstream and 5 nt downstream of the pause
position, consistent with the extent of core nucleic-acid contacts made within the elongation complex (8). To determine the contribution of each
base to pause duration, we used the density of
reads in the NET-seq profile to calculate the relative dwell time of RNAP at each well-transcribed
position in the genome. Modeling the addition
of the next nucleotide as a process with a single
activation barrier, we calculated the effective
energetic barrier to nucleotide addition as the
logarithm of the RNAP occupancy signal (
supplementary materials). We used these values
to determine the sequence dependence of this
1Department of Cellular and Molecular Pharmacology,
Howard Hughes Medical Institute, California Institute for
Quantitative Biosciences, Center for RNA Systems Biology,
University of California, San Francisco, San Francisco, CA
94158, USA. 2Department of Biochemistry, University of
Wisconsin, Madison, WI 53706, USA. 3Department of
Microbiology and Immunology, University of California, San
Francisco, San Francisco, CA 94158, USA. 4Department of
Biological Sciences, Stanford University, Stanford, CA 94025,
USA. 5Department of Applied Physics; Stanford University,
Stanford, CA 94025, USA. 6Department of Genetics,
Stanford University, Stanford, CA 94025, USA. 7Department
of Bacteriology, University of Wisconsin, Madison, WI
*Corresponding author. E-mail: firstname.lastname@example.org (W.J.G.);
email@example.com (R.L.); firstname.lastname@example.org