that enable bacterial communication. Interestingly,
some CARF-domain proteins are not associated
with type III CRISPR-Cas systems, raising a question of how the cOA signaling molecule is generated
in these cases (18). As described here, signaling
pathways involving cOAs (i) provide an additional
level of control for the antiviral defense system
(21), potentially inducing dormancy to buy time
for the host to destroy the invader or promote
programmed cell death of the host (28); (ii) ensure a mechanism for signal amplification; and
(iii) allow robust discrimination from other signaling pathways in the cell.
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We thank A. Rukšėnaitė and L. Taujenis (Thermofisher Scientific)
for help with HPLC-MS experiments; A. Silanskas for HPLC
purification of adenylates; P. Moller Martensen (Aarhus University)
for a kind gift of pOAS1 and pPDE12 plasmids; P. Horvath
(DuPont) for a kind gift of S. thermophilus DGCC8004 strain
and discussions; and S. E. Halford for critical reading of the
manuscript. G. T. acknowledges support from the Research
Council of Lithuania (grant APP-3/2016). M.K., G.K., G. T., and
V.S. are inventors on patent applications related to the work
described in this manuscript: V.S., M.K., and G. T. on international
patent application PCT/IB2015/056756 submitted by Vilnius
University, which covers the StCsm complex and its use
for directed RNA cleavage, and V.S., M.K., G.K., and G. T. on
U.S. provisional patent application 62/512868 submitted by Vilnius
University, which covers production of cyclic oligoadenylates and
their use as allosteric regulators. Expression plasmids used in
this work are available from G. T., M.K., or V.S. under a material
transfer agreement with Vilnius University.
Materials and Methods
Figs. S1 to S24
8 June 2017; accepted 22 June 2017
Published online 29 June 2017
Structure of histone-based chromatin
Francesca Mattiroli,1 Sudipta Bhattacharyya,2*† Pamela N. Dyer,1 Alison E. White,1
Kathleen Sandman,3 Brett W. Burkhart,2 Kyle R. Byrne,2 Thomas Lee,1 Natalie G. Ahn,1
Thomas J. Santangelo,2,4 John N. Reeve,3 Karolin Luger1,4,5‡
Small basic proteins present in most Archaea share a common ancestor with the eukaryotic
core histones. We report the crystal structure of an archaeal histone-DNA complex. DNA wraps
around an extended polymer, formed by archaeal histone homodimers, in a quasi-continuous
superhelix with the same geometry as DNA in the eukaryotic nucleosome. Substitutions of a
conserved glycine at the interface of adjacent protein layers destabilize archaeal chromatin,
reduce growth rate, and impair transcription regulation, confirming the biological importance of
the polymeric structure. Our data establish that the histone-based mechanism of DNA
compaction predates the nucleosome, illuminating the origin of the nucleosome.
The nucleosome consists of two (H2A-H2B) and two (H3-H4) histone heterodimers as- sembled as an octamer that wraps 147 base pairs (bp) of DNA in 1.65 negative super- helical turns (1). Histones, the most conserved proteins known, all have a central “histone
fold” (HF) dimerization motif formed by three a
helices separated by two short loops (fig. S1A).
Small HF-containing proteins, present in most
Archaea, likely share a common ancestor with the
eukaryotic histones (2–4). Hundreds of different
archaeal histone sequences are now known [fig. S1B;
(5, 6)]. Most are 70 ± 5 amino acids long and lack
HF extensions and the basic histone tails, which
are the segments specific to each eukaryotic histone that contribute to nucleosome stability
and gene regulation [fig. S1A; (3, 7)]. Unlike the
mandatory eukaryotic histone heterodimer partnerships, archaeal histones homodimerize and
form heterodimers with related paralogs. Here
we report the structure of archaeal histone-based chromatin and its participation in gene
To obtain crystals, we used a DNA sequence to
fervidus [(HMfB)2] bind at defined locations (8, 9).
In the 4 Å crystal structure (table S1), this 90-bp
DNA wraps around three (HMfB)2 dimers (Fig. 1A)
that are virtually identical when compared to each
other, to (HMfB)2 dimers in the absence of DNA
[(10); root mean square deviation (RMSD) 0.36 Å],
and to the HFs of eukaryotic (H3-H4) and (H2A-
H2B) heterodimers (RMSD ~1.7 Å; Fig. 1, A and B,
and fig. S2A). Each HF dimer (HFD) interacts with
the DNA in a very similar fashion to the eukaryotic
HFDs, with fully conserved amino acid side-chain
interactions [RT pair and RD clamp (R, arginine;
T, threonine; D, aspartic acid); Fig. 1 and fig. S2,
A and B] that mutagenesis studies have confirmed
are essential for DNA binding by HMfB (11, 12).
Intramolecular hydrogen bonds between the two
histones in the (HMfB)2 dimer position the a1 helices
and N termini for optimal interaction with DNA
and direct an N-terminal extension appropriately
through the gyres of the surrounding DNA, as seen
in H2A and H3 in the nucleosome (fig. S2C) (7).
The (HMfB)2 dimers are symmetric and, in the
crystal lattice, polymerize through identical four–a
helix bundles (4HBs; Fig. 2A) to form a contin-
uous helical ramp (Fig. 2B). The geometry of the
4HB is conserved between HMfB-HMfB′, H3-H3′,
and H4-H2B (Fig. 2A), and therefore, the arrange-
ment of any four consecutive archaeal HFDs in
the crystal structure is markedly similar to the
assembly of the four HFDs in the nucleosome
octamer (RMSD 2.0 Å, Fig. 2C). The surface of
the complex formed by archaeal histones has,
however, less positive charge (Fig. 2D).
In the crystal lattice, DNA wraps around the
HMfB protein assembly in a quasi-continuous superhelix, through annealing of the 2–nucleotide
(nt) 5′ overhangs (Fig. 2E). The geometry, diameter,
1Department of Chemistry and Biochemistry, University of
Colorado Boulder, Boulder, CO 80309, USA. 2Department of
Biochemistry and Molecular Biology, Colorado State
University, Fort Collins, CO 80523, USA. 3Department of
Microbiology, Ohio State University, Columbus, OH 43210,
USA. 4Institute for Genome Architecture and Function,
Colorado State University, Fort Collins, CO 80523, USA.
5Howard Hughes Medical Institute, University of Colorado
Boulder, Boulder, CO 80309, USA.
*These authors contributed equally to this work.
†Present address: Department of Molecular Biosciences, University
of Texas at Austin, Austin, TX 78712, USA.
‡Corresponding author. Email: firstname.lastname@example.org