Biology Reference
In-Depth Information
activity. For example, pRNAs
I
-
j´
and
J
-
i´
were inactive in DNA pack-
aging alone, but when mixed together they restored DNA packaging
activity. This result can be explained by the trans-complementarily of
pRNA loops, i.e. the right-hand loop
I
of pRNA
I
-
j´
could pair with the
left-hand loop
a
´ of pRNA
J
-
i´
. Since mixing two inactive pRNAs with
interlocking loops, such as when pRNA
I
-
j
´ and
J
-
i´
are mixed in a 1:1
molar ratio, resulted in production of infectious virions, the stoichiom-
etry of the pRNA is predicted to be a multiple of two (Table 4).
Together with the results from binomial distribution and serial dilution
analyses, it has been confirmed that the stoichiometry of pRNA is six.
One Set Composed of Three Engineered pRNAs:
Multiple of 3
Another set of mutants is composed of three pRNAs,
J
-
p´
,
P
-
k´
, and
K
-
j´
. This set is expected geometrically to be able to form a 3-, 6-, 9-,
or 12-mer ring that carries each of the three mutants. Each individual
pRNA and even mixing any two of the three mutants shows little or
no activity. However, mixing all three pRNAs are with 1:1:1 ratio
restores DNA packaging activity (Table 4). The lack of activity in mix-
tures of only two mutant pRNAs and the restored activity in mixtures
of three mutant pRNAs is expected since the mutation in each pRNA
are engineered in such a way that only the presence of all three
pRNAs will produce a closed ring. The fact that the three inactive
pRNAs are fully active when mixed together suggests that the num-
ber of pRNAs in the DNA packaging complex is a multiple of three,
in addition to being a multiple of two. Thus, the number of pRNAs
required for DNA packaging is a common multiple of two and three,
which is six (or 12, but this number has been excluded by the
approach of binomial distribution (see Section 1(a)).
One Set Composed of Six Engineered pRNAs:
Multiple of 6
DNA packaging activity is also achieved by mixing six mutant pRNAs,
each being inactive when used alone (Table 4). Thus, an interlocking
