Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2014 Jul 17;158(2):314-326.
doi: 10.1016/j.cell.2014.05.038.

Mechanism of transcriptional bursting in bacteria

Shasha Chong  1 , Chongyi Chen  2 , Hao Ge  3 , X Sunney Xie  4
Affiliations

Mechanism of transcriptional bursting in bacteria

Shasha Chong et al. Cell. .

Abstract

Transcription of highly expressed genes has been shown to occur in stochastic bursts. But the origin of such ubiquitous phenomenon has not been understood. Here, we present the mechanism in bacteria. We developed a high-throughput, in vitro, single-molecule assay to follow transcription on individual DNA templates in real time. We showed that positive supercoiling buildup on a DNA segment by transcription slows down transcription elongation and eventually stops transcription initiation. Transcription can be resumed upon gyrase binding to the DNA segment. Furthermore, using single-cell mRNA counting fluorescence in situ hybridization (FISH), we found that duty cycles of transcriptional bursting depend on the intracellular gyrase concentration. Together, these findings prove that transcriptional bursting of highly expressed genes in bacteria is primarily caused by reversible gyrase dissociation from and rebinding to a DNA segment, changing the supercoiling level of the segment.

PubMed Disclaimer

Figures

Figure 1
Figure 1. Transcription on topologically isolated chromosomal DNA loops
(A) Gyrase releases positive supercoiling generated by transcription on a DNA loop, and RNApol keeps transcribing the gene. (B) In the absence of gyrase, active transcription on a DNA loop leads to positive supercoiling accumulation, which inhibits further transcription on the particular DNA loop.
Figure 2
Figure 2. In vitro single-molecule assay to monitor real-time transcription on individual DNA templates using SYTO RNASelect stain
(A) Schematic representation of the experimental arrangement (not drawn to scale). In the presence of 250 nM SYTO RNASelect, nascent RNAs are fluorescent under TIRF excitation at 488 nm. With an excitation power density of 0.22 W/cm2 and an image acquisition time of 5 s, a transcript of 2.3k nucleotides yields a SNR of 1. (B) Fluorescence emission spectra of SYTO RNASelect solution under 488 nm excitation. The dye selectively stains RNA and emits fluorescence with a peak at 530 nm. In the absence of nucleic acids, the dye is not fluorescent at 530 nm. (C) Time-lapse images of 1.1 μm×1.1 μm sub-field-of-view to monitor T7 transcription on one 12 kb-long template. (D) Intensity versus time trajectory of the DNA template shown in (C). Full transcripts are produced repetitively on the template, with transcription elongation time T1 = 500 s and T2 = 300 s, respectively. See also Figure S1 and S2.
Figure 3
Figure 3. Supercoiling dependence of transcription elongation rate
(A) In vitro transcription template design containing a T7 or T7A1 promoter and a 12 kb transcribing sequence. The template is anchored to the flow cell surface via either a single or multiple biotin-streptavidin linkages. (B) Histogram of T7 transcription elongation time on the templates anchored with single (red curve) or multiple biotin-streptavidin linkages (blue curve). The average elongation time for the multiple-biotin template is 60% longer. (C) Titration of T7 transcription elongation rate (23 °C) on the multiple-biotin template (the three bars on the left) with gyrase. The elongation rate increases with the gyrase concentration till it gets as high as that on the single-biotin template (the 4th bar). The elongation rate on the single-biotin template does not change in the presence of a saturating concentration of gyrase (the bar on the right). (D) E. coli transcription elongation rate (37 °C) and T7 transcription elongation rate (23 °C) on the multiple-biotin template are slower than on the single-biotin template. See also Figure S1 and S2.
Figure 4
Figure 4. Supercoiling dependence of transcription initiation rate
(A) Schematic of transcription on a circular template in the absence of topoisomerases. Positive and negative supercoiling annihilate each other after RNApol completes transcription and dissociates from the template. (B) Time dependence of T7 transcription initiation rate under the condition of (A). (C) Schematic of transcription on the circular template in the presence of 41 nM Topo I and 0.1 μM gyrase (same as State 1 in Figure 7A). (D) Time dependence of T7 transcription initiation rate under the condition of (C). The arrow shows the time when the topoisomerases were added into the system. (E) Schematic of transcription on the circular template in the presence of 41 nM Topo I and absence of gyrase. Positive supercoiling is built up as transcripts are produced. (F) Time dependence of T7 transcription initiation rate under the condition of (E). B, D&F are the total intensity versus time from 160 circular templates under respective conditions normalized to the same fluorescence intensity. (G) Time dependence of E. coli transcription initiation rate in the presence of 62 nM Topo I and absence of gyrase. This is the intensity averaged from 106 circular templates at each time point normalized to that from 209 linear templates (Extended Experimental Procedures). See also Figure S1, S2, S3, S4 and S5.
Figure 5
Figure 5. Transition from gene “off” to “on” state
(A) Schematic of transcription on the circular template first in the presence of 41 nM Topo I only (same as Figure 4E), and then both 41 nM Topo I and 0.1 μM gyrase (same as Figure 4C). (B) Time dependence of T7 transcription initiation rate (blue) under the condition of (A). This is the intensity averaged from 160 circular templates at each time point normalized to that from 120 linear templates. Gyrase was added into the system at T = 0, when transcription initiation was essentially stopped by positive supercoiling accumulation. The trajectory after T = 0 is fitted with a single exponential function (magenta). See also Figure S1, S2 and S3.
Figure 6
Figure 6. Transcription processivity and elongation rate upon gyrase inhibition in live E. coli cells
(A) Quantitative RT-PCR measurement of the abundance of different parts of lac operon mRNA under fully induced condition. x-axis: the position of probing sites along lac operon; y-axis: mRNA abundance. Black squares: gyrase partial inhibition by 50 ng/μL novobiocin; Red dots: gyrase complete inhibition by 10 ng/μL norfloxacin. The result indicates non-stop transcription elongation upon positive supercoiling buildup on the DNA. The abundance of each mRNA part is normalized to its abundance under wild type condition, which is plotted as the flat curve. (B) Six sites on lac operon mRNA that were probed in the measurement of transcription elongation rate. (C) 500 ng/μL rifampicin was added into the cell culture at time zero to stop transcription initiation but not elongation. The abundance of different positions on the lac operon mRNA was probed by quantitative RT-PCR at multiple time points. (D) Transcription elongation rate decreased upon gyrase inhibition by 10 ng/μL norfloxacin in live E. coli cells. See also Figure S6.
Figure 7
Figure 7. Dependence of on/off duty cycle ratio (β/α) on effective intracellular gyrase concentration
(A) Kinetic scheme of the two-state model with relevant rate constants. (B) Fitting of cellular ThrS mRNA copy number distribution with “Poisson with zero spike” distribution. (C) Schematics of interactions between effective gyrase concentration and DNA supercoiling generated by transcription under different conditions. Upon gyrase inhibition, positive supercoiling accumulates on the chromosomal DNA loop to a higher extent than wild type. Gyrase overexpression or SGS insertion is the opposite. In a plasmid-borne expression module, positive and negative supercoiling annihilate each other due to the lack of topological barriers. (D) β/α of fully induced lac operon decreases upon gyrase partial inhibition by 50 ng/μL novobiocin treatment, increases upon gyrase overexpression, SGS insertion, and in a plasmid-borne system. (E) β/α of fully induced lac operon and other 18 highly transcribed E. coli genes. β/α of all the 19 genes decrease upon gyrase partial inhibition by 50 ng/μL novobiocin treatment. See also Figure S6, S7 and Table S1.
See this image and copyright information in PMC

Comment in

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Abbondanzieri EA, Greenleaf WJ, Shaevitz JW, Landick R, Block SM. Direct observation of base-pair stepping by RNA polymerase. Nature. 2005;438:460–465. - PMC - PubMed
    1. Bai L, Santangelo TJ, Wang MD. Single-molecule analysis of RNA polymerase transcription. Annu Rev Biophys Biomol Struct. 2006;35:343–360. - PubMed
    1. Baker TA, Funnell BE, Kornberg A. Helicase action of dnaB protein during replication from the Escherichia coli chromosomal origin in vitro. J Biol Chem. 1987;262:6877–6885. - PubMed
    1. Billingsley DJ, Bonass WA, Crampton N, Kirkham J, Thomson NH. Single-molecule studies of DNA transcription using atomic force microscopy. Phys Biol. 2012;9:021001. - PubMed
    1. Blake WJ, M KA, Cantor CR, Collins JJ. Noise in eukaryotic gene expression. Nature. 2003;422:633–637. - PubMed

Publication types

MeSH terms