sample is physically larger, any mechanical errors
in post-expansion sectioning, or stage drift, are
divided by the expansion factor.
The performance of ExM suggests that despite statistical fluctuations in polymer chain
length at the molecular scale, at the nanoscale
distances here examined these fluctuations average out, yielding isotropy. Estimates of mesh
size for comparable gels suggest that the distance between nearest-neighbor polymer chains
are in the ~1 to 2 nm range (17, 18). By tuning
the material properties of the ExM polymer, such
as the density of cross-links, yet higher effective
resolutions may be possible.
REFERENCES AND NOTES
1. T. Tanaka et al., Phys. Rev. Lett. 45, 1636–1639 (1980).
2. I. Ohmine, J. Chem. Phys. 77, 5725 (1982).
3. F. L. Buchholz, Superabsorbent Polymers 573, 27–38 (1994).
4. B. Huang, S. A. Jones, B. Brandenburg, X. Zhuang, Nat.
Methods 5, 1047–1052 (2008).
5. E. H. Rego et al., Proc. Natl. Acad. Sci. U.S.A. 109, E135–E143
6. M. Bates, B. Huang, G. T. Dempsey, X. Zhuang, Science 317,
7. N. Olivier, D. Keller, P. Gönczy, S. Manley, PLOS One 8, (2013).
8. R. W. Cole, T. Jinadasa, C. M. Brown, Nat. Protoc. 6, 1929–1941
9. G. Feng et al., Neuron 28, 41–51 (2000).
10. A. Dani, B. Huang, J. Bergan, C. Dulac, X. Zhuang, Neuron 68,
11. A. Rollenhagen, J. H. R. Lübke, Front. Synaptic Neurosci. 2,
12. G. R. Newman, B. Jasani, E. D. Williams, Histochem. J. 15,
13. K. D. Micheva, S. J. Smith, Neuron 55, 25–36 (2007).
14. B.-C. Chen et al., Science 346, 1257998 (2014).
15. J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, E. H. K. Stelzer,
Science 305, 1007–1009 (2004).
16. J. H. Lee et al., Science 343, 1360–1363 (2014).
17. A. M. Hecht, R. Duplessix, E. Geissler, Macromolecules 18,
18. D. Calvet, J. Y. Wong, S. Giasson, Macromolecules 37,
E.S.B. was funded by NIH Director’s Pioneer Award 1DP1NS087724
and NIH Director’s Transformative Research Award 1R01MH103910-01,
the New York Stem Cell Foundation-Robertson Investigator Award, the
MIT Center for Brains, Minds, and Machines NSF CCF-1231216,
Jeremy and Joyce Wertheimer, Google, NSF CAREER Award CBET
1053233, the MIT Synthetic Intelligence Project, the MIT Media
Lab, the MIT McGovern Institute, and the MIT Neurotechnology
Fund. F.C. was funded by an NSF Graduate Fellowship. P. W. T.
was funded by a Fannie and John Hertz Graduate Fellowship. Confocal
imaging was performed in the W. M. Keck Facility for Biological
Imaging at the Whitehead Institute for Biomedical Research.
Deltavision OMX SR-SIM imaging was performed at the Koch Institute
Swanson Biotechnology Center imaging core. We acknowledge
W. Salmon and E. Vasile for assistance with confocal and SR-SIM
imaging. We acknowledge N. Pak for assistance with perfusions.
We also acknowledge, for helpful discussions, B. Chow,
A. Marblestone, G. Church, P. So, S. Manalis, J.-B. Chang,
J. Enriquez, I. Gupta, M. Kardar, and A. Wissner-Gross. The
authors have applied for a patent on the technology, assigned
to MIT (U.S. Provisional Application 61943035). The order of
co–first author names was determined by a coin toss. The
imaging and other data reported in the paper are hosted by MIT
Materials and Methods
Figs. S1 to S5
Tables S1 to S4
Movies S1 to S3
18 August 2014; accepted 26 November 2014
Replication-transcription switch in
Karen Agaronyan, Yaroslav I. Morozov, Michael Anikin, Dmitry Temiakov*
Coordinated replication and expression of the mitochondrial genome is critical for metabolically
active cells during various stages of development. However, it is not known whether replication
and transcription can occur simultaneously without interfering with each other and whether
mitochondrial DNA copy number can be regulated by the transcription machinery. We found that
interaction of human transcription elongation factor TEFM with mitochondrial RNA polymerase
and nascent transcript prevents the generation of replication primers and increases
transcription processivity and thereby serves as a molecular switch between replication and
transcription, which appear to be mutually exclusive processes in mitochondria. TEFM may
allow mitochondria to increase transcription rates and, as a consequence, respiration and
adenosine triphosphate production without the need to replicate mitochondrial DNA, as has
been observed during spermatogenesis and the early stages of embryogenesis.
The maternally inherited circular mitochon- drial DNA (mtDNA) encodes subunits of complexes of the oxidative phosphoryla- tion chain, as well as transfer RNAs (tRNAs) and ribosomal RNAs (1, 2). Transcription of
human mtDNA is directed by two promoters, the
LSP (light-strand promoter) and the HSP (
heavy-strand promoter) located in opposing mtDNA
strands, which results in two almost-genome-sized polycistronic transcripts that undergo extensive processing before polyadenylation and
translation (3, 4). Note that transcription terminates prematurely about 120 base pairs (bp)
downstream of LSP at a vertebrate-conserved
G-rich region, called conserved sequence block
II (CSBII), as a result of formation of a hybrid
G-quadruplex between nascent RNA and the
nontemplate strand of DNA (5–7). This termination event occurs near the origin of replication
of the heavy strand (oriH) (8) and generates a
replication primer. According to the asymmetric
model (9), replication then proceeds through about
two-thirds of the mtDNA, until the oriL sequence
in the opposing strand becomes single stranded
and forms a hairpin structure. The oriL hairpin is
then recognized by mitochondrial RNA polymerase
(mtRNAP), which primes replication of the light
strand (10). Because replication of mtDNA coincides with transcription in time and space, collisions
between transcription and replication machineries are inevitable and, similarly to bacterial and
eukaryotic systems, likely have detrimental effects
on mtDNA gene expression (11).
We analyzed the effects of a mitochondrial
transcription elongation factor, TEFM, recently
described by Minczuk and colleagues (12), on
transcription of mtDNA. This protein was pulled
down from mitochondrial lysates via mtRNAP
and was found to stimulate nonspecific transcrip-
tion on promoterless DNA; however, its effect
on promoter-driven transcription had not been
determined (12). We found that in the presence
of TEFM, mtRNAP efficiently transcribes through
CSBII (Fig. 1, A and B). Thus, TEFM acts as a fac-
tor that prevents termination at CSBII and syn-
thesis of a primer for mtDNA polymerase. We
identified the exact location of the termination
point in CSBII (fig. S1). MtRNAP terminates at
the end of a U6 sequence (positions 287 to 283
in mtDNA), 16 to 18 nucleotides (nt) downstream
of the G-quadruplex (Fig. 1A). At this point, the
9-bp RNA-DNA hybrid in the elongation com-
plex (EC) is extremely weak, as it is composed of
only A-U and T-A pairs. This is reminiscent of
intrinsic termination signals in prokaryotes—
where the formation of an RNA hairpin is thought
to disrupt the upstream region of the RNA-DNA
hybrid—and is followed by the run of six to eight
uridine 5′-monophosphate residues that further
destabilizes the complex (5, 13).
Human mtDNA is highly polymorphic in the
CSBII region; coincidently, the reference mitochondrial genome (Cambridge) contains a rare
polymorphism in the G-quadruplex—namely,
G5AG7—whereas the majority of mtDNAs from
various haplogroups have two additional G residues (G6AG8) (14). We found that the termination
efficiency of mtRNAP was substantially lower at
G5AG7-CSBII (Fig. 1C), which suggested an effect
of G run length on quadruplex formation and
underscored the importance of further studies of
various polymorphisms in this region.
In considering a putative mechanism of TEFM
antitermination activity, we investigated whether it can interact with the nascent transcript and,
thus, interfere with the formation of the quadruplex structure. We assembled ECs on a nucleic
acid scaffold containing a photoreactive analog
of uridine, 4-thio-uridine, 13 nt downstream from
the 3′ end of RNA, and walked mtRNAP along the
template by incorporation of appropriate substrate
nucleoside triphosphates (NTPs) (Fig. 2A). We observed efficient cross-linking between TEFM and
RNA when the photoreactive base was 15 to 16 bp
away from the 3′ end of RNA. Additionally, using
a template DNA containing the LSP promoter and
Department of Cell Biology, School of Osteopathic Medicine,
Rowan University, 2 Medical Center Drive, Stratford, NJ
*Corresponding author. E-mail: email@example.com