Page 83 - MI-1-1

Microbes & Immunity An ATP-free packaging of T4 DNA

A B C

Figure 6. A comparison between on-filter-wet and on-filter-dry DNase degradations. After 2 days of treatments, on-filter-wet treatment did not degrade
virions (A and B), while on-filter-dry treatment digested almost all virion particles, but small blobs of DNA remained (B and C). The brightness of images
was inverted for visuality. Scale bar: 5 μm.
In this study, the initial concentration of ATP molecules ATP dependence in the former and independence
3,32
in dialyzed virion suspension before packaging was ca. in the latter; and motor molecule dependence for DNA
20 pM (Figure 3), which is more than 1000 times lower packaging in the former, taking several minutes to package
than the minimum ATP concentration for packaging DNA one DNA into a capsid. In the latter, a group of packaging
41
in the motor-ATP system (25 μM ATP). The packaging processes happen in parallel and in the ensemble average,
5
of DNA molecules into capsids occurred at the condition ca. 80% of packagings might have been done within 3 min
of up to 10 -fold dilution from this concentration of (data not shown). Another difference between the two
7
ATP; 10 -fold dilution for ejection of DNA in 30 mM systems is that the ratios of the regenerated virions were
2
Pi solution, following 10 -fold dilution with 30 mM ca. 10% in the former, while the efficiencies in the latter
3
42
solution plus 10 -fold dilution with EL at the compaction were >90% in both FLM and pfu counts (Figure 1). Besides
2
process (column IV of Table 2). There was practically no these differences, the motor-ATP system has a limitation
ATP at the compaction/packaging process of DNA. This for working as the sole packaging system of DNA in a
indicates that the packaging system of DNA into a capsid cell. As globular DNA cannot be packaged into capsids,
introduced here is not ATP-dependent and not a motor- packaging of DNA should be initiated when they are in
ATP system. The sole agent causing the ejection and a coiled state under high intracellular Pi concentration.
packaging of DNA in this protocol was the change of the On the other hand, packaged DNA flows out of a capsid
ambient Pi concentrations. The DNA molecules in the in the high Pi condition. 8-10 Indeed, if the packaging
capsids of T4 were first ejected by increasing the ambient process is interrupted by the addition of ATPγS during the
concentration of Pi. Without addition of multivalent transportation process, the packaged DNA would run out
cations at valences higher than three, 38,39 simply decreasing from the capsid. This indicates that during the packaging
43
the ambient concentration of Pi induced the fluid-to-solid process, once the pumping activity of the motor stops, the
conformational change of DNA. This compaction of DNA DNA molecule will automatically run out from the capsid,
occurs inside a capsid, which is confirmed by the resistance and complete virions cannot stay in a cell. The stabilization
of the compacted DNA particles against DNase I and the of the complete virions requires a lower concentration
ability of infection (Figure 1). The efficiency of packaging of Pi, which induces DNA coils to undergo the globular
to form the infective virion is nearly 100% (Figure 1). This conformational change (Figures 1, 2, and 4), 14,15 and
process does not require additional ATP for packaging of disables packaging by the ATP-motor system. This can be
DNA. The concentration of the ambient ATP indicates that an obstacle if the ATP-motor system is the only packaging
the process is an ATP-free system. However, the process process. On the other hand, in the conformational change
itself includes the change of the ambient concentrations system, once the free ends of DNA molecules are connected,
of Pi, implying the differences of the concentrations of or partially packaged into capsids spontaneously or by
37
ambient ions or the density gradient energy as the energy the ATP-motor system, 3,32,37 the decrease of the chelate
source for the ejection and packaging processes. This type (Pi) concentration induces the packaging of DNA (in this
40
of packaging system is hereinafter called “conformational study), which is followed by the spontaneous joining of
change system.” head and tail to form infectious virions. 32,33 Accordingly,
Characteristic differences between the motor-ATP the sole motor-ATP system or sole conformational change
system and the conformational change system are as follows: system may not be able to accomplish the packaging of

Volume 1 Issue 1 (2024) 77 doi: 10.36922/mi.2666