All dsDNA viruses multiply their genome and assemble
a procapsid, a protein shell devoid of DNA. The genome
is subsequently inserted into the procapsid. The bacterial
virus phi29 DNA translocating motor contains a hexameric
RNA complex composed of six pRNAs. Recently, we found that
pRNA dimers are building blocks of pRNA hexamers. Here,
we report the structural probing of pRNA monomers and dimers
by chemical modification under native conditions and in
the presence or absence of Mg2+. The chemical-modification
pattern of the monomer is compared to that of the dimer.
The data strongly support the previous secondary-structure
prediction of the pRNA concerning the single-stranded areas,
including three loops and seven bulges. However, discrepancies
between the modification patterns of two predicted helical
regions suggest the presence of more complicated, higher-order
structure in these areas. It was found that dimers were
formed via hand-in-hand and head-to-head contact, as the
interacting sequence of the right and left loops and all
bases in the head loop were protected from chemical modification.
Cryoatomic force microscopy revealed that the monomer displayed
a check-mark shape and the dimer exhibited an elongated
shape. The dimer was twice as long as the monomer. Direct
observation of the shape and measurement of size and thickness
of the images strongly support the conclusion from chemical
modification concerning the head-to-head contact in dimer
formation. Our results also suggest that the role for Mg2+
in pRNA folding is to generate a proper configuration for
the right and head loops, which play key roles in this
symmetrical head-to-head organization. This explains why
Mg2+ plays a critical role in pRNA dimer formation,
procapsid binding, and phi29 DNA packaging.