REVIEWS
The DNA-packaging nanomotor
of tailed bacteriophages
Sherwood R. Casjens
Abstract | Tailed bacteriophages use nanomotors, or molecular machines that convert
chemical energy into physical movement of molecules, to insert their double-stranded DNA
genomes into virus particles. These viral nanomotors are powered by ATP hydrolysis and pump
the DNA into a preformed protein container called a procapsid. As a result, the virions contain
very highly compacted chromosomes. Here, I review recent progress in obtaining structural
information for virions, procapsids and the individual motor protein components, and discuss
single-molecule in vitro packaging reactions, which have yielded important new information
about the mechanism by which these powerful molecular machines translocate DNA.
Tailed bacteriophages
Double-stranded-DNA
bacteriophages with a protein
tail that attaches to a
susceptible bacterium. DNA is
injected into the host through
the tail.
Prophages
Bacteriophage genomes that
are physically integrated into
the chromosome of the host
bacterium.
Nanomotor
A molecular motor that
functions on a nanometre scale
to convert energy into directed
physical movement of
molecules.
Procapsid
The preformed protein
container into which DNA is
packaged during virion
assembly.
Division of Microbiology and
Immunology, Pathology
Department, University of
Utah School of Medicine,
15 North Medical Drive East,
Salt Lake City, Utah 84112,
USA. e‑mail: sherwood.casjens@
path.utah.edu
doi:10.1038/nrmicro2632
There are many different types of bacteriophages (viruses
that infect bacteria). Those that have proteinaceous tail
structures and double-stranded DNA (dsDNA) genomes
— the tailed bacteriophages — are the most abundant
‘life form’ in Earth’s biosphere, with at least 1030 extant
free virus particles, about ten for each bacterial cell on
Earth1–5. Such numbers strongly imply that tailed bacteriophages are critically important in the ecology of our
planet. In addition, the fact that many bacterial virulence
genes reside in prophages (summarized in REFS 6–8) and
the potential for the eradication of disease-causing bacteria through the use of bacteriophages (bacteriophage
therapy)9,10 make these viruses relevant to human health.
As they are easily manipulated in the laboratory, these
phages had a central role in the early investigations of
genes and how genes function. The lytic life cycles
of bacteriophages, which typically take about 1 hour per
generation and produce 50–500 progeny, are reasonably
well understood (FIG. 1a). In addition, many phages have
an alternative lysogenic life cycle during which almost
none of their genes are expressed and their genome
becomes part of the genome of the host bacterium,
where it can reside quiescently for many host generations. One currently exciting area of research concerning
these viruses is the study of the nanomotor that trans­
locates the dsDNA chromosome into the virion during
the assembly of progeny phage particles in the lytic
growth cycle (FIG. 1b).
The mechanism by which dsDNA is packaged into
tailed bacteriophage virions has fascinated molecular
biologists since it was realized, over four decades ago,
that these structures contain long (19–500 kb) dsDNA
molecules that are hundreds of times more compact than
dsDNA in solution. Experiments in the 1970s showed
that, rather than condensing the DNA first and then
assembling a shell around this DNA core, tailed-phage
DNA is inserted into a preformed protein container
(called a prohead or procapsid)11,12. Many molecular
genetics studies since then have indicated that the basic
machinery of dsDNA packaging is similar in all tailed
phages. Escherichia coli phages λ, P1, P2, T3, T4 and T7,
Salmonella enterica phage P22 and Bacillus subtilis phages
ϕ29 and SPP1 have been studied in the most detail13.
There are three proteins or functions that are universally
required for assembly of the procapsid, and two for packaging DNA into this structure13–18. The three procapsid
proteins are the coat protein and the scaffolding protein,
which assemble the icosahedral shell of the procapsid,
and the portal protein, which forms a dodecameric ring
at the ‘portal vertex’ of the procapsid (FIG. 1b). DNA enters
and leaves through this unique vertex during packaging and injection, and tails complete virion assembly by
attaching at this vertex after DNA is packaged. The scaffolding protein exits the procapsid before or during DNA
insertion and so is not present in the completed virion.
The two packaging proteins recognize DNA, thread it
through the portal ring and pump it into the procapsid
(FIG. 1b). There are variations in the details of this theme;
for example, phage T4 has a second capsid shell protein
at the icosahedral vertices as well as multiple scaffolding
proteins; E. coli phage HK97 combines scaffolding and
coat functions into one protein; and phage ϕ29 has one
packaging protein and one packaging RNA (see below).
Work towards identifying the components and
understanding the function of this molecular motor proceeded at a steady pace for 30 years, but the first insights
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a
Portal vertex
into the detailed mechanism of the packaging motor
have now been gained from recent breakthroughs in the
determination of the structures of participating proteins,
procapsids and virions, as well as single-molecule DNApackaging experimental systems. In addition to the
ecological and medical importance of bacteriophages,
their potential applications in nanotechnology make it
imperative that these molecular motors are understood
in as much detail as possible. This Review summarizes
recent progress in our understanding of the operation of
this motor; the reader is referred to previous reviews for
historical details13,14,16–18.
heads and the lengths of the viral DNA chromosomes
that are contained within them indicates this compaction; for example, the 14,000‑nm-long chromosome of
phage λ must fit into its 60‑nm-diameter head. Results
from low-angle X‑ray scattering indicate that DNA within
the head is nearly as compact as crystalline DNA15, so
there is little room for DNA-binding or compaction
proteins between the double strands and, at least in the
simpler tailed phages (such as ϕ29, λ and P22), there
are no essential genes that are candidates for encoding
such proteins. However, it is energetically unfavourable
to place dsDNA into such a configuration, owing to
charge repulsion, DNA bending and other factors19–22,
so it is inevitable that DNA is forced into the procapsid by
an energy-dependent motor. Cryo-electron microscopy
has been used to generate three-dimensional reconstructions of several tailed-phage virions, and these
reconstructions indicate that the DNA is arranged
within the virion as an imperfect solenoid, the central axis of which is parallel to the tail axis23–35 (FIG. 2).
Similar DNA loops have been observed in individual
virions36,37. In the reconstructions, the DNA is most
clearly defined (that is, most similarly positioned in
all individual virions) near the interior surface of the
coat shell and near the portal vertex. In some virions a
cylinder of protein electron density extends into the
head at the portal vertex 25,26,38–40, and it is possible to
imagine that the DNA is oriented and held in place in
part by the proteins that reside there. The innate stiffness of DNA and the crowded conditions may help
keep the chromosome from becoming completely
disordered after packaging.
Early studies indicated that DNA is usually packaged in one direction from a particular starting point
and that the last end packaged is the first end out during
injection15, although phage T4 and its relatives may be
an exception, with the first end in also being the first
end out 41. Virions often appear to have electron density
within the DNA entry–exit portal channel, and this may
be due to the presence of DNA at this location, poised
for release during injection25,26,29. DNA release is not discussed in detail here, but if the DNA is in a high-energy
state in the virion, then at least the first portion of the
DNA should spontaneously eject from the virion when
the plug in the tail is removed following binding to a
cellular receptor, and this can happen in vitro in some
cases19,42,43. The suggestion that such a high-energy
state is important seems to be a reasonable hypothesis
and is not without theoretical support (at least in the
final stages of head filling), but it is called into question
by the facts that osmotic pressure within cells opposes
DNA injection from phage particles44 and transcription is required to ‘pull’ the DNA from the virions of
some phages (most notably, T7) into infected cells45,46.
Current studies are aimed at understanding these
apparent paradoxes.
The unique vertex to which the
portal protein and tails are
attached. Icosahedral
structures such as phage heads
have 12 five‑fold rotationally
symmetrical vertices.
DNA organization in tailed-phage virions
The product of genome packaging in tailed phages is a
dsDNA molecule that is highly compacted within the
viral capsid. Simple observation of the sizes of phage
DNA-packaging motor components
The molecular motor that pumps the DNA into tailed
phage procapsids usually consists of three proteins: the
portal protein and two DNA-packaging proteins (and, in
Genomic dsDNA
Host bacterium
Circle-to-circle
replication
Rolling circle
replication
Protein
synthesis
DNA packaging
Procapsids
Viral
proteins
Heads
with
DNA
Tails
b
Bacteriophage head
DNA movement
Portal ring
Portal protein
TerL
TerS
DNA-packaging motor
Figure 1 | Tailed bacteriophages and their DNA-packaging motor. a | The major
Reviews
Microbiology
steps in the lytic life cycle of a typical tailed bacteriophageNature
are shown
from|left
to right,
with emphasis on virion assembly. The adsorption of a virion to a cell surface receptor is
followed by injection of the linear double-stranded DNA (dsDNA) genome, which then
adopts a circular form. The early and late viral genes are expressed, leading to the
synthesis of viral proteins. Replication of viral DNA takes place through a circle-to-circle
mechanism followed by a rolling-circle mechanism in many phages. The viral proteins
assemble to form procapsids and tails, and then the viral DNA is packaged into the
procapsids, resulting in DNA-containing heads. Finally, heads and tails join to form
complete virions, which are released after cell lysis. b | A highly simplified depiction of
the DNA-packaging motor, showing the location of the dodecameric portal ring at one
vertex of the icosahedral bacteriophage head. This portal ring is bound to the terminase:
the packaging ATPase (large terminase subunit (TerL)) and DNA recognition protein
(small terminase subunit (TerS)) are shown as rings of five and eight subunits, respectively;
however, these numbers may not be the same in all phages. The nature of the
attachments between these three motor components is not known, and the physical
arrangement shown is speculative, especially in terms of the position of TerS. The black
arrow represents the location of the dsDNA during motor action and the direction of
DNA movement.
Portal protein
The part of the DNA-packaging
nanomotor that forms the hole
or portal through the phage
capsid; DNA enters and exits
the virion through this portal.
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a
b
c
Coat protein
(gp5)
DNA
Portal protein
(gp1)
gp4
gp10
Tail spike
protein (gp9)
Tail needle protein (gp26)
Figure 2 | Arrangement of DNA and the portal protein in bacteriophage P22
Nature Reviews | Microbiology
virions. a | A cross section (~60‑Å thick) through the centre of the asymmetrical,
three-dimensional reconstruction (derived from cryo-electron microscopy) of the P22
virion at 7.8 Å resolution, coloured to highlight individual components: portal protein
gp1, ‘head completion’ proteins gp4 and gp10, coat protein gp5, tail spike protein gp9,
tail needle protein gp26 and DNA. b | A cutaway view of the P22 virion (tilted slightly
toward the viewer) at 17 Å resolution, showing the two outer DNA layers. c | The outer
layer of the intra-virion P22 DNA (tilted slightly away form the viewer), extracted from
the structure in part b. Image courtesy of J. Johnson, The Scripps Research Institute,
La Jolla, California, USA. Scale bar is 200 Å. Part a image is reproduced, with permission,
from REF. 40 © (2011) Cell Press. Part b image is reproduced, with permission, from
REF. 26 © (2006) American Association for the Advancement of Science.
Low-angle X‑ray scattering
A technique that is used to
determine the radially
averaged electron density of
particles that make up an
unoriented (as opposed to
crystalline) sample. It is based
on the deflection of a beam of
X-rays away from its straight
trajectory after it interacts with
the particles in the sample.
Terminase
The DNA nanomotor protein
complex that recognizes the
DNA which will be packaged.
The terminase contains the
ATPase activity that converts
chemical energy into
mechanical movement, and
also contains the active site for
cleavage of double-stranded
DNA (when such a site is
present).
Concatemers
Long DNA molecules that
contain multiple head-to-tail
repeats of the phage genome
sequence. Concatemers are
often formed as the result of
the DNA synthesis machinery
replicating around a circular
template multiple times
without terminating.
one case, an RNA molecule is required in addition; see
below) (FIG. 1b). These phage proteins seem to be highly
divergent47,48, and in many comparisons the proteins that
perform these functions have no recognizable amino
acid sequence similarity (other than the Walker A/Blike ATP-binding motifs in the large terminase subunits)
with their counterparts from other phages. However, the
structural similarities between the motor proteins across
diverse phages (discussed below) suggest that tailed
phages ‘invented’ the dsDNA-packaging apparatus once
in the long-distant past and, although they have evolved
differences in the components, they probably still
all use a similar underlying mechanism. The protein
components of the motor are described below.
The portal ring. DNA is transferred into the procapsid
shell through a ring of portal proteins (called connector
proteins in some systems) that lies at the portal vertex
of the procapsid. Portal proteins are present in procapsids and virions as a dodecameric ring in all phages for
which they have been examined (λ, P22, T4, T3, T7,
SPP1, P2 and ϕ29)26,30,38,49-54. Crystal structures are now
available for portal proteins from phages ϕ29, SPP1
and P22 (REFS 55–57), and the similar polypeptide folds
of their central domains indicate that they are highly
diverged homologues in spite of the fact that they have
little amino acid sequence similarity. The portal ring
has a channel at the centre, through which DNA enters
the virion during packaging and exits during ejection
(FIGS 2–4). The numerous DNA-binding proteins that
are present in host cells are not packaged into tailedphage virions; presumably, their absence is ensured by
the passage of DNA through the portal channel, which
is too narrow to easily accommodate bound proteins.
The portal ring forms the site on procapsids to which
the rest of the packaging motor, as well as eventually the
head completion proteins and the tail, attaches. In certain systems, the portal proteins can exist in several
different conformations58,59, but the role of such conformational flexibility is not known. The central channel of the portal ring is wide enough to allow passage
of a single dsDNA molecule, but flexible polypeptide
loops that extend into the channel in some cases must
move out of the way during packaging 59. The reasons
for the large differences in the size of portal proteins
(for example, 309 amino acids in ϕ29 and 725 amino
acids in P22) are not understood, but the largest portal
protein for which the structure is known, gp1 of phage
P22, has a long carboxy‑terminal extension that resides
in the interior of the virion40,57 (FIGS 2,3). The role of this
extension is not known, but it has been suggested that
it facilitates genome spooling within the interior of the
capsid or, like a rifle barrel, increases the accuracy of
genome ejection into the host cell. No ATP-binding or
ATP cleavage site has been found in portal proteins.
The dodecameric portal ring replaces a pentamer of
coat proteins at the portal vertex of the procapsid. This
means that the interaction between the coat and the portal proteins is complex, because 12 molecules of portal
protein interact with five molecules of coat protein. This
interaction has been visualized at subnanometre resolution in several phages25,26,29,30 and in P22 procapsids60;
however, the details of exactly how the ring is held in
place remain unclear. In addition, the scaffolding protein
is often required for successful incorporation of the portal ring into the virion, and the P22 scaffolding protein,
gp8, has substantial interactions with both coat and portal in the procapsid60–62 (FIG. 4). Although it was initially
proposed that the portal ring rotated relative to the coat
as part of a possible packaging mechanism63, in fact the
ring does not seem to rotate during packaging or in
the completed virion26,64,65.
Curiously, in phage ϕ29 and its relatives, five molecules of a 174‑nucleotide (nt)-long phage-encoded
RNA molecule, called pRNA, are present at the portal
vertex of the procapsid54,66. The secondary structure of
pRNA is essential for packaging, and although the exact
role of pRNA is not yet known, it is required for assembly of the ATPase part of the motor and is released after
packaging 66–68.
DNA recognition, cleavage and translocation. Many
tailed phages use DNA replication mechanisms that
produce head-to-tail concatemers of the phage genome
sequence. In these cases, the packaging machinery
includes an integrated nuclease activity that cuts individual chromosomes from the DNA concatemer. The
results of such cleavages are molecules that contain precisely one genome sequence (with several different types
of ends, depending on the phage system), genomes with
specific direct terminal repeats, or circularly permutated genomes with a low percentage of terminal redundancy 69. After an initial cleavage of the substrate DNA,
one of the two ends thus created is threaded through the
motor and into the procapsid; DNA is then pumped into
the procapsid, and a second DNA cleavage occurs only
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a TerL from phage T4
b TerS from phage Sf6
c Portal protein from phage P22
d Portal protein from phage P22
(C)
C
N
29 aa
DNA movement
ATP-binding site
N
N
C
Figure 3 | Structures of the three DNA-packaging motor proteins. a | A monomer (610 amino acids) of the large
terminase subunit (TerL) from phage T4 (REF. 81). The amino‑terminal ATPase and carboxy‑terminal
are
Nature nuclease
Reviews |domains
Microbiology
coloured green and orange, respectively, and the ATP-binding site resides in the cleft indicated by the black arrow171.
b | Side and top views of the homo-octameric small terminase subunit (TerS) from phage Sf6 (REF. 101). Alternate identical
subunits (each of 140 amino acids) are shown in blue and yellow. c | Side and top views of the portal protein gp1
dodecamer from phage P22. Alternate identical subunits (each of 725 amino acids) are shown in red and green. d | A side
view of the portal protein gp1 monomer from phage P22 (REF. 57). The full length of the C-terminal helix, which extends
for another 20 helical turns (see part c), is not shown. A section of 29 flexible amino acids that is not seen in the crystal
structure is denoted ‘29 aa’ here. The black arrow marks the central channel and points in the direction of DNA movement
during packaging. Parts are not drawn to scale. The ribbon diagrams were created with MacPyMOL (Schrödinger LLC,
New York, USA).
after the head is full of DNA. A subsequent packaging
event can then preferentially begin at the unpackaged
DNA end that was created by the previous cleavage, to
give rise to a processive series of packaging events70–72.
Again, phage ϕ29 and its relatives are an exception: their
dsDNA replicates as linear, unit-length molecules with a
protein covalently attached to both 5′-ends, so no nuclease
is required73.
The two packaging proteins are usually called the
large and small terminase subunits. This nomenclature derives from the phage λ proteins that were first
shown to be required for formation of the termini of
the packaged DNA74. Although the term terminase
remains in use, these proteins are now known to also
recognize DNA for packaging and to be the translocase
that moves DNA into the procapsid (see below). The
large and small terminase subunits are referred to here
as TerL and TerS, respectively, for brevity. Biochemical
analyses have shown that in some cases (for example,
for P22) TerS and TerL are found in phage-infected
cells as a complex 75 (H. R. Brown and S.C., unpublished
observations), whereas in other cases (for example, for
T4) they do not seem to be tightly bound to each other 76.
TerL is monomeric when expressed alone77–80 and consists of two major domains81 (FIG. 3a). The amino‑terminal
domain of TerL contains a Walker A/B motif, which is
often found in proteins that bind and cleave ATP82,83.
Biochemical analyses of TerL homologues from phages
ϕ29, λ, P22, T3, SPP1 and T4 (REFS 77,84–88), and cocrystals of the T4 TerL (also known as gp17) with ATP81,
confirm this ATPase activity. Cleavage of ATP to ADP
at this active site almost certainly generates the energy
required for DNA packaging, as genetic modifications
that affect this ATPase activity have commensurate
effects on the packaging reaction87,89–92. The C‑terminal
domain of TerL contains the nuclease active site that
cleaves DNA during packaging 93–95, and it has a similar
polypeptide fold in phages T4 and SPP1 (REFS 81,94).
In the phages for which it has been studied, TerS
is responsible for recognition of the DNA that will be
packaged. In phage λ, TerS (also known as gpNu1)
binds several specific sites near the cos site (the
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a
b
Coat proteins
Portal protein ring
Putative
scaffoldingprotein density
Figure 4 | The procapsid of phage P22. a | A view of the portal vertex of an asymmetrical,
Nature Reviews
| Microbiology
three-dimensional cryo-electron microscopy-based reconstruction
of the P22
procapsid
at 8.7 Å resolution. The coat proteins are blue and green, depending on the distance from
the centre of the particle, and the portal ring is purple. The coat protein hexamers that
surround the portal vertex are outlined by black ovals. b | A central slice though the
procapsid. Yellow represents the inside surface of the coat protein shell, and red
represents putative scaffolding-protein density. Figure is modified, with permission,
from REF. 60 © (2011) US National Academy of Sciences.
Occam’s razor
A line of reasoning that argues
that the simplest explanation
should be favoured until a
more complex one is required
by the observed data
(attributed to the Franciscan
friar Father William of Ockham).
sequence-specific nuclease cleavage site). The recognition site (called pac) for P22 TerS (also known as
gp3) lies within the ~120‑bp region where a sequenceindependent packaging initiation cleavage occurs (see
below). TerS is required for packaging initiation in vivo,
but it is not yet clear whether it has any role in DNA
translocation. It is not required for the functioning
of the phage T4 packaging motor in in vitro experiments76,96. However, in other cases TerL and TerS form
a stable complex, so it is possible that TerS is present on
the motor during DNA translocation in those cases97.
The oligomeric state of phage λ TerS varies with solution conditions, but the N‑terminal domain is dimeric
and the whole protein forms larger oligomers98,99. The
P22 TerS is a nonamer 100, and the TerS (also known as
G1P or gp1) from the Shigella flexneri phage Sf6 is an
octamer 101 (FIG. 2). In phages T4 and SPP1, TerS (also
known as gp16 and G1P, respectively) is probably also
an octamer 102,103. This diversity in subunit number, as
well as the apparent ability of the P22 TerS to also use
rings of nine or ten subunits77, suggests that the spatial
organization of TerS is less constrained than that of the
other two motor proteins. TerS is also more variable in
amino acid sequence than the other two motor proteins104. The phage Sf6 TerS and an N‑terminal fragment of the phage λ TerS have similar folds98,101, but
until full structures are obtained for other TerS proteins
we will not know whether these are highly diverged
homologues or the result of convergent evolution. The
genome of phage ϕ29 does not encode any TerS protein.
DNA-packaging motor action
Over the past four decades, many cleverly designed
models have been proposed to explain the molecular mechanism of the motor that packages DNA into
tailed-phage procapsids. Most of these have been tested
and found wanting, and these have been reviewed elsewhere16,105. Nearly all researchers in this field are now
in agreement that DNA packaging is powered by an
ATP-hydrolysing DNA translocase106; some argue that
electrostatic interactions within the virion and/or conformational changes of the capsid may also contribute
to the energetics of packaging 107,108. As the site for ATP
binding and cleavage resides in the N‑terminal portion of TerL, it has been proposed that the terminase
itself physically moves the DNA in response to ATP
hydrolysis. However, it has also been hypothesized that
the energy from ATP cleavage is transmitted from the
terminase to the portal protein, which in turn moves
the DNA. Occam’s razor would seem to favour the former
model, but neither has been rigorously tested and ruled
out. Collectively, the many in vivo molecular genetics
studies and in vitro DNA-packaging reactions that have
been carried out have determined the protein components and biochemical requirements for packaging.
These experiments have universally indicated that the
virally encoded portal and terminase proteins are sufficient to form the basic translocase motor without help
from host proteins (although host proteins can have
accessory roles; see below).
The stoichiometry of the proteins in the packaging motor remains unclear; all motors examined have
a dodecameric portal ring, and the phage ϕ29 and T4
TerL proteins (also known as gp16 and gp17, respectively) that are assembled onto the portal vertex of the
procapsid have electron densities that are consistent with
five such subunits being present 54,81,109. FIGURE 5 shows a
three-dimensional reconstruction, from cryo-electron
microscopy, of the phage T4 procapsid with bound TerL.
Although it has not yet been proved directly that five
TerL molecules are bound to the portal vertex, these
studies present a good argument that this is the case81
(although in solution the phage λ terminase forms a
ring-shaped TerL–TerS complex that contains only four
TerL molecules110). The location and number of TerS
molecules in the assembled motor is not known for any
phage, but the different oligomeric states of the various
TerS proteins in isolation suggest that this number may
vary depending on the phage.
Bulk solution measurements using in vitro packaging
reactions have concluded that about 2 bp are packaged
per ATP cleaved by the motors of phages ϕ29 and T3
(REFS 84,86). The energy derived from this number of
ATP cleavages is calculated to be sufficient to account
for the amount of energy that is required to compact
DNA to intra-virion density 111,112. Recent optical tweezer
single-molecule experiments pioneered by Bustamante
and colleagues (BOX 1) have shed considerable light on
the functioning of the DNA-packaging motor. This technology has been applied to the motors of phages ϕ29,
λ and T4, and some of the properties of these motors
are shown in TABLE 1 . Interestingly, the maximum
rates of DNA translocation (180–1,800 bp per second)
appear to correlate with the length of DNA that is packaged by these phages, which suggests that perhaps the
motor does not work any faster than it needs to for the
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a
b
Portal protein
200 Å
dsDNA
c
ATPase domain of TerL
Nuclease domain of TerL
Figure 5 | DNA-packaging motor of phage T4. a | A three-dimensional
cryo-electron
Nature Reviews | Microbiology
microscopy reconstruction of the phage T4 procapsid with bound subunits of large
terminase subunit (TerL). The coat protein (gp23) is shown in white; vertex protein (gp24)
is magenta; small outer capsid protein (Soc) is green; highly immunogenic outer capsid
protein (Hoc) is yellow; and the bound TerL appears as two rings coloured orange and
blue. b | A magnification of the portal vertex from part a. The white areas represent
electron density, in which ribbon diagrams for TerL and the portal protein are modelled.
The ribbon diagrams are: red, the phage SPP1 portal protein gp6 (which is used here as a
model for the T4 portal protein, the structure of which has not yet been elucidated); blue,
the carboxy‑terminal nuclease domain of T4 TerL; and yellow and green, subdomains I
and II of the amino‑terminal ATPase portion of T4 TerL (subdomain I contains the
ATP-binding site). c | The indicated ring of electron density in panel b is reoriented 90°
to show a view along the central channel. In parts b and c, a scaled double-stranded
DNA (dsDNA) molecule is shown. Image is modified, with permission, from REF. 81
© (2008) Cell Press.
individual phages. The rates determined in this way
agree reasonably well with other estimates; phage P22
and phage λ DNA is packaged at a rate of about 500 bp
per second in cell extracts113,114, and T4 DNA is packaged at a rate of about 900 bp per second in vivo41,115. The
speed of translocation is affected by the load, so as packaging fills the head and it becomes more difficult to push
more DNA into the capsid, the speed decreases116–118
Box 1 | Single-molecule analysis of DNA packaging using optical tweezers
Single-molecule studies of DNA
packaging utilize the attachment of a
single actively packaging procapsid–
motor–DNA complex to two
microspheres, with the procapsid shell
on one sphere and the distal end of the
DNA being packaged on the other
(see the figure). Both microspheres
are captured in optical traps (the
narrowest point of a focused laser
beam). The solution conditions can
be varied, and the distance between
the two spheres can be observed
and manipulated by moving the
microspheres with the optical tweezers
to study the biochemical requirements,
translocation speed, force generated
and step size of the DNA-packaging
motor112,116–119. Figure is modified, with
permission, from REF. 16 © (2007)
Elsevier.
Fixed
microsphere
DNA
Motor
movement
DNA-packaging
motor
Procapsid
Movable
microsphere
(analogous to a spring becoming more difficult to compress the further it is compressed). The overall time it
takes to package a DNA molecule must also take into
account the motor pauses and backwards slips that are
observed in such reactions. The mean length of active
translocation without a slip reflects the ‘processivity’
of the motor, which differs somewhat among the three
phages (TABLE 1). Although their operating speeds may
vary, the three motors studied can apply similar maximum forces of 60–110 piconewtons112,116–118. This force
is sufficient to fill phage heads with dsDNA to the density that is universally found in such virions (TABLE 1).
These results indicate that the nanomotor that packages
dsDNA into tailed phages is one of the strongest known
molecular motors and can apply about eight times the
force of the kinesin motor, 20 times that of the skeletal muscle myosin molecular motor and about twice
that of E. coli RNA polymerase (calculated in REF. 112).
If backwards slippage is ignored, about 30% of the
energy from ATP cleavage seems to be converted into
physical motion of the DNA 18,111,112, and the ‘power
density’ (power generated / motor volume) of the phage
packaging motor is about twice that of an automobile
engine117.
The details of how the DNA-packaging motor physically translocates DNA are the target of current investigations. Careful analysis of the phage ϕ29 motor with
optical tweezers yielded the surprising finding that
packaging occurs in steps of 10 bp, and that each of
these steps comprises four smaller substeps of 2.5 bp119.
Although these substeps have not been unequivocally
proved to correspond to individual ATP cleavage events,
it seems likely that this is the case, given that about 2 bp
are packaged per ATP cleavage. This substep size is likely
to be universal if all packaging motors have the same
mechanism; however, it is important that these analyses
are extended to other phage motors that have unique features. For example, in ϕ29, the packaging ATPase (TerL)
is exceptionally small (332 amino acids, compared with
610 amino acids for the T4 TerL), there is no apparent
TerS, and the pRNA and terminal protein (gp3; which
is covalently bound to the DNA ends) are required for
packaging; in theory, either of pRNA or gp3 could provide TerS function. The non-integral number of base
pairs in each substep (2.5 bp) and the four substeps
within each larger step put important constraints on
physical models of motor function. These observations
require coordination among the several TerL molecules
in the motor so that four subunits bind ATP and then
sequentially ‘fire’ in rapid succession to move the DNA
in bursts of four 2.5‑bp movements119. The power stroke
of the motor has been argued to be generated by product release after ATP cleavage111,119–121, and the subunit
coordination might be mediated by an arginine finger
motif that is present in this type of ATPase120. Studies
with mutant TerL proteins are beginning to discover
which parts of the protein are involved in force generation and motor speed91,92. Models for motor action are
complicated by observations which suggest that five TerL
subunits (rather than four, which would correspond to
the four substeps) appear to be bound to ϕ29 and T4
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Table 1 | Properties of DNA-packaging molecular motors
Phage
Native DNA
length (kb)
Density of packaged
DNA (bp per 105 Å3)*
Processivity
(kb per slip)‡§
Packaging speed
(kb per second)§||
Motor force
(piconewtons)§
ϕ29
19.3
46
>15
180
110
λ
48.5
47
>45
700
>60
T4
166
48
~13
1,800
>60
*Densities are calculated in REF. 172. ‡During packaging, the DNA can slip back out of the procapsid a little. These values give the
average length of DNA that is packaged between slips. §See REFS 111,112,116–118,120,173. ||The maximum rate with no
opposing force (that is, at low capsid fullness and saturating ATP concentration); the rate decreases with increasing load.
IHF
procapsids54,81,109 and that DNA may be physically compressed during insertion122. Several hypotheses have
been put forward for how these motors might physically
move DNA within some of these constraints, and they
involve, for example, flexing between the two domains
of TerL81, a molecular lever in each TerL subunit that
pushes the DNA120, or a ‘lock washer’-type arrangement
of TerL subunits that moves the DNA by interconverting
between a spiral and flat ring 119.
The above picture of the DNA-packaging motor is
certainly oversimplified. For example, there may be a
second ATP-binding site in the C‑terminal nuclease
domain of TerL123; the TerL nuclease activity is affected
by the presence of the N‑terminal ATPase domain, ATP
and TerS94,124–127; the TerL ATPase activity is affected by
DNA and TerS126,127; and TerS subunits may also bind
and cleave ATP128. Whether the DNA-binding sites for
the nuclease activity and for DNA translocation are the
same or different also remains unclear 93. The DNAtemplate requirements of the motor have been studied,
and it has been found that the ϕ29 motor can efficiently
package across a region of 10 bp of methylphosphonate DNA (which is uncharged) embedded in normal
dsDNA, but the packaging efficiency drops dramatically
if the uncharged region spans 11 bp. If only the 3′‑to‑5′
strand (in the direction of packaging) is uncharged,
at least 30 bp of such hybrid DNA can be packaged by the motor, whereas this length cannot be packaged
if the 5′‑to‑3′ strand is uncharged. Surprisingly, the
motor can successfully package normal dsDNA that
contains 10 nt in each strand for which the opposing
bases cannot pair, a 20‑nt single-stranded DNA gap, a
10‑nt single-stranded DNA bulge or even a non-DNA
double-stranded linker, and the force dependence of
these reactions indicates that the modified regions
are not simply getting through the motor channel by
diffusion129. Nicks (that is, breaks in one of the DNA
strands) are tolerated by the T4 motor, except for in
substrates that are longer than 500 bp, in which nicks
block packaging quite efficiently 130. Finally, although
all known tailed-phage virions contain only one large
DNA molecule, the T4 packaging motor can package
multiple smaller DNA molecules into a single procapsid
in vitro96,131.
(Integration host factor). An
Escherichia coli protein that
binds DNA and bends it at a
sharp angle. IHF was first
discovered as a cofactor of the
phage λ enzyme integrase.
Control of the motor and the accessory functions
Recognition of DNA, accessory factors and motor
assembly. How the DNA to be packaged is recognized
and cleaved varies among the tailed phages. The spatial
Late-gene transcription
factors
The phage-encoded early
proteins (which are expressed
early during infection) that
cause transcription of the
genes which are expressed at
late times after infection.
relationship between the packaging recognition site
and the DNA cleavage site is an example of such differences. Phage λ TerS binds to several sites located
about 100‑bp downstream (in the direction of packaging) of the sequence-specific cleavage site98,132; for
P22, an initiation cleavage occurs without sequence
specificity within a region of about 120 bp surrounding the packaging recognition site133; and for phage Mu,
the packaging recognition site is near one end of the
integrated phage genome (the substrate for packaging in
this phage), and the cleavage that initiates packaging
occurs without sequence specificity in the adjacent host
DNA, 150±70‑bp away from the recognition site134–136.
Furthermore, at least some headful packaging motors
appear to be able to diffuse substantial distances along
the DNA after DNA recognition and before the cleavage event that initiates insertion of DNA into the procapsid137. Variations in amino acid sequence among
TerL proteins correlate with their different cleavage and
packaging initiation strategies69,138. In addition, in phage
T4, late-gene transcription factors are involved in the
initiation of packaging in a manner that is not yet
completely understood 139; in phage T7, the phageencoded RNA polymerase is involved in the initiation of packaging 140; and in T7 and SPO1, the DNA
polymerase appears to be involved in duplication of the
chromosomal terminal repeat during packaging 141,142.
Finally, IHF, a DNA-binding protein of the E. coli host,
is involved in the recognition of substrate DNA for
phage λ packaging 143.
The portal ring is a structural part of the procapsid
and virion, but TerS and TerL are not typically found
bound to the procapsid or the completed virion. The
details of the assembly of TerS and TerL onto the portal ring to form the complete packaging motor are not
well understood. In some phages, the data are consistent with a model in which TerS binds DNA and TerL,
and this terminase–DNA complex in turn binds to the
procapsid portal ring through its TerL subunits144,145.
In phages λ, T3 and P22, genetic analyses suggest that
procapsid–TerL interaction is mediated by a region of
TerL near its C terminus104,146,147. However, there are variations on this scheme in other phages, as the TerL proteins of ϕ29, SPP1 and T4 can bind their cognate portal
proteins (as isolated proteins or in procapsids) without
DNA or TerS under in vitro conditions 54,117,118,148,149, and
sequences outside the C terminus have been implicated
in the binding of T4 TerL to the procapsid149 (S. Hegde
and V. Rao, personal communication).
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Termination of packaging and motor disassembly. After
the phage chromosome is fully inserted into the procapsid, DNA cleavage releases the packaged DNA from the
concatemer (again, phage ϕ29 is an exception, as there is
no packaging-associated cleavage). This cleavage can be
sequence specific, as for the so-called ‘cos’ phages such as
phage λ, for which staggered nicks are made in the DNA
to create the two cohesive ends of the virion chromosome. Alternatively, for the so-called headful-packaging
phages such as P22, Mu and T4, cleavage occurs without
sequence specificity when the head is full of DNA69. Both
cos phages and headful-packaging phages have a mechanism that recognizes when the DNA within the capsid
has reached ‘virion density’ and signals the nuclease portion of the terminase to cleave the DNA. For phages P22
and SPP1, the portal protein is an important part of this
‘headful-sensing device’ (REFS 150,151). For phage λ, for
which headful cleavage occurs only at a specific nucleotide sequence (the cos site), there is an additional DNA
site (cosQ) that the motor must pass over in order to
license such a cleavage152. Models have been suggested
for the headful-sensing mechanism, such as a slowing
of the rate of DNA translocation as the head fills145, the
requirement of the motor to apply more force as the head
fills145, and sensing internal DNA density according to
DNA proximity to the portal protein in the virion interior 26 (portal proteins can exist in more than one conformation58,59). However, none of these models has yet been
proved to reflect the actual mechanism.
After packaging is complete, the terminase is released
from the nascent virion. Terminase release is poorly
understood, but if, after the headful cleavage, the terminase remains associated with the end of the DNA that
was not packaged, it could be responsible for initiating
the next packaging event in a packaging series on a concatemer. The proteins that bind the portal ring to replace
the terminase in phages P22, HK97 and SPP1 have similar folds (despite having little sequence similarity)57,153,154,
suggesting that they have similar functions.
Plugging the channel. Following release of the terminase
proteins, DNA is not stably packaged and will leak out
through the portal vertex channel unless this passage is
plugged. This is perhaps best understood in the shorttailed phage P22, for which the portal vertex channel
is extended by the stepwise addition of two additional
‘head completion’ proteins, gp4 and gp10 (REFS 155,156),
onto the portal ring, and the distal end of the channel is
finally plugged by the tail needle protein gp26, a protein
for which the crystal structure has been determined157.
In long-tailed phages such as phage λ, the tail is added
as a complete unit and can contain the plug, because
(in some cases at least) one end of the packaged DNA
enters some distance into the tail channel in the completed virion158–160. However, in phage SPP1, the portal
channel seems to be plugged in the virion by a specific
protein (gp16)161; this plug protein is widely conserved
among the viruses that are closely related to SPP1 (longtailed phages with non-contractile tails), suggesting
that this mechanism may be common in this group of
phages154. Such plug proteins must get out of the way
during DNA ejection to allow DNA passage through
the portal and the tail channels. The phage P22 plug is
released from the virion during injection162, whereas the
phage SPP1 plug is opened by a conformational change in
response to a signal that is sent from the tip of its long tail
upon receptor engagement 154,161. After terminase release
and the binding of the plug and ‘head completion’ proteins, tails join to the head spontaneously to form the
complete virion.
Strengthening the capsid shell. In some phages, shell
assembly is accompanied by the formation of covalent
isopeptide bonds that crosslink coat subunits, resulting in a ‘chainmail’ of interlocking rings163. In addition,
many tailed phages encode proteins that bind to the
outer surface of the head and strengthen the capsid after
the coat protein shell is assembled. Some of these proteins — such as Dec of phage L164,165, and small outer capsid protein (Soc) and highly immunogenic outer capsid
protein (Hoc) of T4 (REFS 166,167) — are dispensable
under normal conditions (FIG. 5a). However, the analogous protein of phage λ, head decor­ation protein gpD, is
normally essential; in the absence of gpD, capsids containing full-length chromosomes burst from the pressure
of the internal DNA116,168. All these proteins bind to the
outer surface of the head only after the coat protein of
the procapsid has undergone the conformational change
that results in shell expansion before or during DNA
packaging. This conformational change is not reviewed
here because there is currently no strong evidence that
it affects the DNA-packaging motor directly; the expansion can happen before packag­ing in phage T4 and
appears to occur during packaging in phage λ.
Concluding remarks
The recent burst of structural progress concerning
phage DNA-packaging proteins, as well as the introduction of optical tweezer technology into this field,
has allowed the formulation of much more detailed
ideas for the mechanism by which the DNA-packaging
motor pumps DNA into phage procapsids. However,
many questions remain about the stoichiometry and
precise spatial relationships of the two terminase
proteins in the assembled motor, about the detailed
mechanism of force generation and the resulting DNA
movement, about the functional interactions between
different parts of the motor, about the mechanism of
DNA recognition and threading into the motor in vivo,
about the connection between capsid fullness and the
termination of packaging, and about the mechanism of
motor disassembly after the termination of packaging.
These issues are being addressed experimentally, and
we hope to have the answers in the near future. In the
meantime, potential applications of packaging nano­
motors in nanotechnology and biology are beginning
to be described. For example, the nanomotors could
be used for efficient delivery of nucleic acids or related
molecules across barriers such as cell membranes or for
applications in single-molecule DNA sequencing 169,170.
There is little doubt that this will be a fertile research
area for some time into the future.
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Acknowledgements
I thank M. Feiss, V. Rao and S. Grimes for thoughtful reading
of this manuscript before publication. My research is supported by the US National Institutes of Health grant RO1
AI074825.
Competing interests statement
The author declares no competing financial interests.
FURTHER INFORMATION
Sherwood R. Casjens’s homepage:
http://www.path.utah.edu/research/cbi/sherwood-casjens/
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