The stability of nucleoprotein complexes at varied ionic strength

Lüe Sciences Vol. 10, Part II, pp. 949-954, 1971. Printed in Great Britain

Pergamon Press

TFE STABILITY OF NUCLEOPROTEIN COMPLEXES AT VN2IED IONIC STRENGTH Iver Agrell Zoophyeiological Irotitute, University of Lund, Lund, 9iwdsn . (Received 2 December 1970; in final form il February 1971)

In earlier im~estigetioro (l, Z) it has boon quantitatively abeesved how ONA end RNA road end canpeta for histone and can form ndxad nucleoprotein ca~plexes under defined conditioro . The present article deals with the detec tion of ouch complexes through msaeunrrant of their stability et varied ionic strength and it will be demorotreted haw RNA and 0Nß mutuslly stabilize eeoh other et nucleoprotein fornetion . Materials end Methods The basic nodal oxparimsnts wee arranged as sirtultaneou mituel psscipitatiaro of DNA, RNA and histone, at the precipitation minimm of 0 .12 M NeCl, using a double gradient system to cover the whole concentration range of the three pen~ters (1, 2) . One double gradient concsrnsd. ONA end RNA and the other nucleic void and historo . The substances used wars high nolecular ONA (Signs, Type I), yeast RNA (Sigma, Type XI) end F2b prvpersd in our laboratory (3) . Precipitatae were produced et seven rvpresentetiw points, within the double gradient system, plotted in Fig. 4. The aarpoaitian of these precipitates is depicted in the text of Fig . 1 . The stability of the precipitated nucleoproteins was examined es their solubility in increasing cancentntioro of NeCl, the axtrnctions being made et 0.2, 0.4, 0 .6, 0.8, 0.9 and 1.0 M NeCl . At 0 .91.0 M NeCl awry precipitate tested dissolved 100 per cent . The ease of extraction should be a nreeurs of the dissociability of the precipitates end thus indicate the psssenca of variously stable cartplexes . All extrectiaro ware made at +4°~ for 12 hours, end wars initiated and termiroted by stirring at roam tarpereture . The extracted amo~nta of histone end nucleio acid wen meesund apectraphotarrotrioelly through differential readings et tivo wewlengths (2) . DNA wee estimated with the diphenylamdrw reaction end the aro~nt of RNA was calculated es the difference . For interpretation, the observed extraction curves mist undergo same kind of canparison . T}w references chosen wars RW1-F2b end ONA-F2b prscipitatsd and extracted separately and thus independently. The follwing exenple 949

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P~r c~nt ~atraet~d

FIG. 1 Extinction calves of mixed

nucleoprotein complexes of varying cosrpasition . The percen-

taga weight csxtpoaition, DNAs RNAsF2b, is the followings A ZOs30s50, 8 ~ 10130 :60, C ~ 35s

too

15s50, D - 22s15s63, E ~ 4116s

60

53, F - 3aI61so . The extrac-

ti m curves for the precipitate

:o

DNAsRNAsF2b ~ 12 :32:56 are not

to

mntal exaes NaCl cancantintion (M) at axtroction . Vertical

included in this figure . hbri-

axed Par cent extracted nucleic acids and histone, logerithmic seals, o - RNA, ~ DNA,

toe

e - F2b . Unbroken linaa~

abservad extinction . Broken linae - expected extraction .

40

20

10

6 7

o

as

M Natl

t o

as

11 NaCI

t

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Stability od Nucleoprotein Complexes

z F o

351

.10

N d

C

.05

10

20

30

40

50

60

Per cent contributory RNA FIG . 2 Stabilization of DNA by RNA. Vertical axis : Difference betrraen the obaeswd and expected DNA extraction cu:we in Fig . U-1F, expressed es M NeCl . Horimntal axis: RNA extracted toSether with DNA as pert cant of total nuddc acid . illwtretea ttse procedure wad to abtein expected pracipitetion conies : At an initial cascentratiort of DW1sFiNAsF2b - 0.19 :0 .58 :0 .25 rtg/ml (nucleic acids F2b - 0.75 :0.25) a pracipitete is obtained canteininQ DNAsRN11sF2b ~ 0.10 :0 .15: 0 .25 mg. Ms;smdng that the te+o nuclaoprotains ara pa~ecipitatad indapandantly. the pracipitete should be rerode up of 0 .10 ~ ONA + 0.10 mg F2b and 0 .15 mQ RW1 " 0 .15 mg F2b, since at the initial concentrations wad the F2b/nucldc acid ratio for both DW1 and RNA nucleoprotein pr9cipiteted separately is about 1 (2). It is consequently expected that the two nucleoproteins will be extracted by NaCl independently of each other . fie observed extraction of DNA and RNA

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Nucleoprotein Complmces

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Per cent extraet~d

SO

FIG . 3 Exenplification of RNA extraction curves, L RNA, subdivided into two fractions . One frbctian is extracted together with ONA at higher ionic strength, stable RNA. The other is extracted alone at lower ionic strw:gth, labile RNA. A Fig. 1F, B ~ Fig. 1B . Vertical axes : per cent extracted . Horizontal axes : NaCl concentration (M) et extraction . respectively from the precipitate is therefore compered with the extraction of separately prepared ONA-F2b end RNA-F2b precipitates made up of equal parts of nucleic acid and F2b . For the histone, the sum of the extraction of F2b from these two nucleoproteins repres~ts its expected extraction . The referancea for the other ONA :RNßsF2b precipitates tested were prepared in a corresponding manner by separate precipitetians of DNA and RNA by F2b in adequate proportions .

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FIG. 4 Type ellotnent of RNA precipitated within the double gradient syatam . The figures denote the percentage amount of RNA of total RNA extracted independently of DNA at lower imic strength . Vertical axial initial ONA-RNA gradient . Horizontal axial initial histone-nucleic acid gradient . The expected extraction curves for each nucleic acid end tFw histone (Fig . 1) nn rather independently of the relative contenta of histone end nucleic acid in the precipitates . ONA- histone is lees easily extractable than jiNA-historw and the DNA-histone of higher F2b/DNA ratios being the most stable . Results and Discussion The extraction curves obtained with nucleoprotein precipitates pr9pered fr~an ONA and RNA combined deviate systematically from thane expected, Fig. 1 . The presence of RNA .makee DNA more difficult to extract end vice versa, RNA extraction from the precipitate obtained et high RNA and histone concentration being an apparent exception, Fig. 18 . In ell cases it was found that uP to about 0 .5 M NaCl only RNA is extrectad . Thin RNA has a tendency to dissolve rtor9 easily than expected . The nucleic acids are always extracted together with hietorw,

and the histone/nucleic acid

ratio of the extracts is independent of the strength of tFw NaCl solution . It can be seen from Fig. 1, that the course of the extraction curve for hietorw shifts from that of an RNA protein to that of a ONA protein . A similar course is described by the extraction curves for RNA end there is a good oorteùtion between the relative artount of RNA extracted togeüwr with DNA end ainulteneoua

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stabilization of 0Nß, Fig . 2 . file mutual stabilization of RNA and ONA et nucleoprvtein formation strongly indicates the appearance of distinctive mixed nuclaopr~otein conplexes possibly build up by aor~ molecular ersnnge~nt bet~wen RNA and DNA consolidated by the histone . Because the stabilization appears at higher ionic strength, electraetatic interactiore should be ndnimel and the stranger binding instead depend more upon conforTnationel factors . fie extraction curves for RNA, and for hietane, can rowdily be subdivided into two frnctiana, es shown in Fig . 3, which also brings Fig .~1B into line with the other extraction curves . One of the RNA fractions is~extracted together with DNA and prabebly through soma linkage to DNA, stabilized es is DNA . The other fraction of RNA is extracted alone at lower ionic strength . fie relative content of these two fracture in the precipitates veriee depending upon the initial RNA end histone concentration, Fig . 4, The two RNA fractions discussed may differ in molecular weight . fie DNA linked fraction may be the more low molecular species, since it has been observed that law molecular RNA can bind firmly to DNA (4) . On the other hand high molecular liver ribosomal RNA has a great capacity to attach to DNA histone at initialhigher RNA erd histone concentrations (2), where the more labile RNA predardnwtea . A strong binding of RNA to histone and DNA hoe earlier been observed especially in rapidly dividing cells (5, 6) . Acknwledgenents-This investigation wes facilitated by grants from the Royal Physiognephical Society . Yeluable technical aid was given by Miss Brite Nilesan . References 1.

I .P .S. AGRELL, Acte Chem, Stand . 24, 1085 (1970) .

2.

I .P .S . At~tELL, Biochim. Biophys . Acte 186, 226 (1969) .

3.

E.W, JOHNS, Biochem . J . 92, 55 (1964) .

4.

M,E . OßF~B and D.J. McC01~BdEll, Biochemistry ~, 1524 (1969) .

5.

I .P .S. AGRELL, Pathol . Biol . Semaine Hop . ~, 284 (1961) .

6.

I .P .S. AGRELL and H.A, BERGQUIST, Biochem . Phyeiol . 22, 1~ (1967) .