Interaction of ribosomes and polydeoxyribonucleotides

Vol. 13, No. 4, 1963

BI~H~MICAL

INACTION

AND BIOPHYSKAL

RESEARCH CO~UNlCATiONS

OF BIBOSOMESAND POL~BO~IBO~CLEOTIDSS

Yituru Takanami* and Toshio Oksmoto Research Institute for Nuclear Medicine and Biology Hiroshima University, Hiroshima, Japan

Received

September

12, 1963

In the course of study to analyze interaction

permitting

the

between messenger ENA and ribosomes, we reached the

conclusion

that

ribosomes could interact

chains in a "freely formation

conditions

rotating*

with

configuration,

polyribonucleotide resulting

in the

of riboaome aggragates(Okamoto 8r Takanami,1963, Similar

nami & Okamoto,l063). laboratories(Barondes

observation

& Nirenberg,l902,

Warner et ai.,l963,

Gierer,1963,

wae made in several

Spyridee

Staehelin

Taka-

& Lipmann,l962,

al 1963, Gilbert, -et --

1903), and the name "polyaome" was proposed for such aggregates (Warner et a1.,1963). action

further,

To elucidate

it was of great

polymer of deoxyribonucleotide8 riboeomes or not.

the mechanism of this

interest

to know whether the

shows similar

affinity

We thus examined the affinity

to the onea used in a previous

toward

of two kinds of

phage DNA, those of 9X174 end T2, under experimental similar

inter-

study(Takenami

conditions 82Okamoto,

1963). Results of the experiment polyribonucleotidee, with ribosomes, A solution K.Mateubara, *

single

clearly

demonstrated

that,

like

strand DNA alone could be associated

forming a heavy complex. of (32F')-+X174 phage was kindly

University

provided

of Kanazawa, Japan. (32p),T2

Present address: Brookhaven National New York. 297

Laboratory,

by Dr,

phage was

Upton, L.I.

BIOCHEMICAL

Vol. 13, No. 4, 1963

prepared

AND BIOPHYSICAL

T2 phage infected

from

E.coli

RESEARCH COMMUNICATIONS

B(B),

which had been ex-

posed to a medi~(Tri~-glucose)

containing

(3~)-pho~phate

about one generation

infection.

DNA was extracted

the respective then treated viscosity.

time after

from

phages by the phenol method. Each DNA solution by ultrasonic

oscilator

for

parations

became acid-soluble

that

little

labeled

of theee pre-

by DNase treatment,

BNA wae present

was

6 min. to reduce its

Since more than 90% of the radioactivity

very

for

it was confirmed

a% a contaminant

in these

preparations. The procedures the technique gradient

for preparing

used for

sedimentation

O.OlM Trie,

p3 7.6,

pB 7.6,

15'C),

analysis

were the aame as those described

(Takauami 61Okamoto,lQSI).

Tris,

ribosomea from E.coli

Approximate

in a previous

About 2 mg of ribosomea,

of the mixture

waa layered

in 5 n&i Yg 2+-

sedimentation

analysis

coefficient

the ratio

the vicinity

was adjusted

on top of a linear

and sedimentation

mated assuming that

paper

and 2 to 10 pg of DNA, in 0.05M NaCl-O.OlM

and Yg2+concentration

gradient(S-20$),

and

in sucrose density

was mixed at a room temperafure(in

Then, the mixture

B(E),

of

to 5 mM.

eucrose density

was carried

out.

of unknown peaks was esti-

of the distance

meniscus by any two components is nearly

traveled

constant

from the

(Martin

& Ames,

1961). First,

the affinity

The sedimentation given

in Fir.1

the radioactivity coefficient distributed conditions the shift with

profile

(a).

of gbxL74 DNA toward ribosomes was tested. of the #Xl74 DNA-ribosome mixture

As can be seen from the figure,

wa% found in a heavy region,

is

almost all

the sedimentation

of the main peak being around 110s. #Xl.74 DNA alone in a region lighter than 70s ribosomes under the same of eeatrifugation. It wae thus strongly snggeeted that of the radioactivity

ribosomee.

It

was noted,

was due to the complex formation however, 298

that,

unlike

the mixture

BIOCHEMICAL

Vol. 13, No. 4, 1963

AND BIOPHYSICAL

(a)

RESEARCH COMMUNICATIONS

(b) F-.+X DNA alOne b

0.4

1200

800 ? I 4 I a 0

% 8 (u 0.2 g

400

5

10

15

5

TUBE

Fig.1

and ribosomes,

more rapidly

sedimenting

solution

might contain

configuration Accordingly,

prevented

approximate

linear

the modified

such a secondary

for 20 h

& Okamoto,1963). under simi.ar

coefficient

When condi-

of the main

was confirmed

$x174 DNA. Assuming that

chain of spherical

by

DNA could form much larger

sedimentation It

stabilized

Z$ formaldehyde

DNA alone gave a sedimentation

of original

an equivalent

with

DNA was tested

peak being around 2lOs(Fia.l(b)). aldehyde-treated

weight

of the polysome structure.

previously(Takanami

of the modified

molecular

RNA, 9x174 DNA in

and that

the formation

it was found that

aggregates,

natural

own strand,

+Xl74 DNA was treated

as described

the affinity

like

some secondary structure its

to form much

than the po1y-U molecule employed.

reason, we assumed that,

hydrogen bonds within

strand DNA failed

complexes, whereas its

For this

to that

NUMBER

the single

was assumed to be much larger

tions,

15

Sedimentation analysis of ribosome-$X174 DNA mixtures. (a) Untreated 'KU74 DNA. Centrifuged at 25,OOOrpm for 3 h. (b) Formaldehyde-treated 9x174 DNA (F-+X DNA). Centrifuged at 25,OOOrpm for 1 h.

of poly-U

at 37'C,

10

that

profile

the formsimilar

the aggregate

is a

70s ribosomes, which is approximated

elipsoid(Gierer,l9B3),

it 299

was estimated

that

the

by

BIOCHEMICAL

Vol. 13, No. 4, 1963

complex consisted original three

It

has been assumed that

reaction

amino groups of bases, involved

about a dissociation

1959). Accordingly, the validity

On the contrary,

$%l74 DNA might be combined with only two or

70s riboaomee.

brings

RESEARCH COMMUNICATIONS

of 9 to 10 ribosome particles.

untreated

aldehyde with

AND BIOPHYSICAL

of form-

in hydrogen bondings,

of secondary structure(Doty

above results

would provide

et al.,

good evidence

for

of our speculation.

(b)

Ia)

(Cl H-F-T2DNA a10lle IV

- 2000

I' I 1 i

0' I

I - 1000

, 5

10

15

5

z P 0

: q

15

10

TUBE NUMBER

Fig.2

Sedimentation analysis of ribosome-T2 DNA mixtures. Untreated T2 DNA. (b) Heated T2 DNA (H-T2 DNA). Heat- and formaldehyde-treated T2 DNA (H-F-T:! DNA). Centrifuged at 25,OOOrpm for 3 h.

Similar

type

of experiments

place of $Xl74 DNA. In contrast

was carried

to &Cl74 DNA, however,

atrand DNA showed no significant Then, a solution

of significant

the heated DNA was treated the quantity

involved

(b),

interaction

further

with

(a)).

5 min. at

the double-stranded such a denatured DNA with

riboeomes. When

2% formaldehyde

in the interaction 300

the double

to ribosomes(Fig.2

in order to abolish

As can be seen in Pig.2

gave an indication

at 37'C,

affinity

T2 DNA in O.OW NaCl was heated for

of

100°C and cooled rapidly, configuration.

out using T2 DNA, in

for

20 h

became much

BlOCHEMlCAL

Vol. 13, No. 4, 1963

larger,

AND BIOPHYSICAL

and more than 50s of the radioactivity

the heavy complex region(Fig.2 In a previous

somes on their test

was transferred

paper, we reported

that

30s subunit

30s subunit(Okamoto

and suggested that

& Takanami,l963),

formed on the mixtures

the

to ribo-

In order to

of DNA toward each ribosome subunit,

kinds of subunits were prepared and sedimentation

and individual

alone could

as messenger DNA, would be attached

the affinity

then,

analysis

As the results,

the denatured DNA showed interaction

two

was per-

of the heat- and formaldehyde-treated

subunits.

to

(cl).

form a complex with polyribonucleotides, pofyribonucleotide,

RESEARCH COMMUNlCATlONS

T2 DNA

it was demonstrated that

with 30s subunit

alone(Fig.3).

(b)

0.4

? i

% 2

1000

0.2

z 0

0”

i: TUBE

Fix.3

It

Sedimentation analysis of mixtures of heatBpa formaldehyde-treated T2 DNA and individual ribosome subunits, (a) 508 subunit. (b) 308 subunit. Centrifuged at 25,000 rpm for 3h.

seems poasible

to conclude,

of deoxyribonucleotides, an ability

like

to associate with

single-stranded

and freely

of a polynucleotide the nature

NUMBER

of its

that

therefore,

of ribonncleotides,

ribosomes, if

rotating

that

form.

301

possesses

the chain exists

in a

In other words, affinity

chain toward ribosomes does not sugar component.

the polymer

depend upon

BIOCHEMICAL

Vol. 13, No. 4, 1963

Still, action.

AND B~OPHYSI~L

we can say little

The fact

that

about the mechanism of this

chain has an important

role

that

phosphate groups,

ration

ia not so flexible

easily

accomodate itself

Institutes

However, we can

the chain is associated

since the double-stranded

upon the surface

Eesearch Funds of the Ministry the National

interaction.

a8 a single-stranded

This work wa8 Supported

with

the base aide of the nucleotide

in this

out the poasibility

somes by its

inter-

the double strand DNA had no affinity

ribosomea appears to suggest that

not rule

RESEARCH CO~UNICATIONS

in part

configu-

one and will

not

of the ribosomes.

by Grants from the Scientific

of Education,

of Health,

to ribo-

U.S.A.

Japan (No.95637),

and

(GM-105&S-01).

Reference8 3aronde8,S.H.

& Nirenberg,M.W.,

Science,

138, 813 (1982)

Doty,P., Boedtgar,H., Freeco,J.B., Haeelkorn,E. Natl, Acad. Sci, U.S., 45, 4S2 (1959) Gierer,A.,

J. Mol.

Biol.,

J. Mol. Biol.,

Martin&G.

& Ames,B.N.,

2, 374 (1963) J. Biol.

& Takanami,M., & Lipmann,F.,

Staehelin,T.,

Wettatein,F,O.

Warner,J.B., 122 (1963)

Knopf,P.M.

Chem,, 238, 1372 (1961)

Biochim.

Spyride8,G.J. (1962)

Takanami,M. & Okamoto,T.,

Proc.

i3, 148 (1963)

Gilbert,W.,

Okamoto,T.

& Litt,Y.,

Proc,

Biophps. Acta, Natl.

L Eoll,E., J. Mol.

Biol.,

& Rich,A.,

Proc.

302

Acad. Sci.

63, 325 (1963) U.S.,

Seietnce, l&&

43, 1971

180 (1903)

in presn. Natl.

Acad. Sci,

U.S.,4&