Page 65 - The Biochemistry of Inorganic Polyphosphates

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Complexes of polyphosphates with nucleic acids 49
Comparison of the peak areas on the electrophoregrams showed, however, that the PolyP
component was much smaller than it should have been had it contained all of the PolyP
present in the complex. Thus, in the less PolyP-rich fraction, instead of a PolyP/RNA ratio
of 1:4, the electrophoregram showed a ratio of 1:11, and electophoregram examination of
a fraction with a PolyP/RNA ratio of 9:1 gave peaks in the ratio of 4:1. This suggested that
some part of the PolyP present in these fractions was combined with the RNA, while the
other part was in the free state.
In order to establish whether the PolyP was bound to the RNA, even if only partially,
by divalent metal cations, the PolyP–RNA fractions were dialysed before electrophoresis
against a 10 −3 M solution of the known complexing agent EDTA. This treatment resulted in
a certain increase in the PolyP peaks, although it did not lead to complete separation of all
PolyP from the RNA. These results suggested that divalent metal cations played some role
in the formation of PolyP–RNA complexes. In order to confirm this assumption, a series of
experiments on separation by electrophoresis of artificial mixtures of PolyP and RNA was
carried out. In these studies, PolyP from a yeast acid-soluble fraction with an average chain
length of 30 residues and synthetic sodium PolyP with an average chain length of 75 residues
were used. The pure RNA material (Merck) was the same in both cases. Examination of
2+
the results showed that the mixtures, which contained yeast PolyP (with Ca ), displayed
marked discrepancies in the PolyP to RNA ratios before and after separation in a Tiselius
apparatus. The area under the early displayed peak was substantially smaller than it should
have been with regard to the initial PolyP content in the material. On the other hand, the
PolyP/RNA ratio remained nearly the same when the mixture of RNA with synthetic PolyP
was analysed. These results demonstrated the absence of covalent bonds in the complexes
isolated from biological material. It is very likely that PolyP and RNA are bound by divalent
metal cations.
It should be noted that in the work of Ebel and co-workers (Ebel et al., 1958c; 1962,
1963; Dirheimer and Ebel, 1964a; Dirheimer et al., 1963) techniques for the preparative
separation of PolyP–RNA complexes from yeast by using activated carbon (Ebel et al.,
1962; Muller-Felter and Ebel, 1962; Stahl and Ebel, 1963; Stahl et al., 1964) and Sephadex
G-200 (Dirheimer and Ebel, 1964a) were developed. In addition, these complexes can
be separated into their components by precipitation of the PolyP in the presence of high
concentration of barium salts (Belozersky and Kulaev, 1970).
Belozersky and Kulaev (1970) and Stahl and Ebel (1963) showed that Ca 2+ and Mg 2+
ions were responsible for the formation of very stable and ‘difficult-to-separate’ PolyP/RNA
complexes. Investigations into the possible existence of covalent or hydrogen bonds in
these complexes have shown that both forms of bonding are absent, while electrostatic
2+
interactions mediated by Ca ,Mg 2+ and other metal ions are possibly present (Ebel
et al., 1962; Belozersky and Kulaev, 1964, 1970). It should be noted that there might
be a certain similarity between RNA–PolyP and PHB–PolyP, in particular, participation of
divalent cations in the linkage of the two polymers. The model of the linkage of the PolyP
and RNA chains through such divalent cations is presented in Figure 4.3.
The question of the functions of RNA–PolyP complexes needs further investigation. It
is probable that the complexing with PolyP enhances the RNA stability. Some evidence
has been obtained that in E. coli PolyPs inhibit RNA degradation by degradosome (Blum
et al., 1997).
The possibility of PolyP interaction with DNA is now confirmed by data evidencing
its participation in gene activity control (Kornberg, 1999; Kornberg et al., 1999). Earlier,