Vol. 4, No. 6, 1961 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

OTHE DEPENDENCE OF CELL-FREE PROTEIN SYNTHESIS IN E. COLI

UPON RNA PREPARED FROM RIBOSOMES,

st

Heinrich Matthaei* and Marshall W. Nirenberg

Laboratory of Biochemistry and Metabolism, National Institute
of Arthritis and Metabolic Diseases, National Institutes of Health,
Bethesda, Maryland
Received March 22, 1961

A stable cell-free system has been obtained from E. coli which incorpor-
ates c 4 evaline into protein at a rapid rate. This system is of particular
interest since certain parameters resemble protein synthesis in intact cells
(1). The purpose of this communication is to describe a novel characteristic
of the system; that is, a requirement for high molecular weight ribosomal RNA,
needed even in the presence of soluble BNA and ribosomes.

Washed E. coli W3100 cells harvested in early log phase were disrupted by
grinding with alumina and enzymes were extracted with 2.5 volumes of buffer
containing 0.01 M Tris, pH 7.8, 0.01 M Mg acetate, 0.06 M KCl, 0.006 M mercapto-
ethanol (standard buffer). The extract was centrifuged at 20,000 x g for 20
minutes; supernatant fluid was decanted and 3 yg DNAase per ml was added to
reduce its viscosity. The supernatant fluid was centrifuged again at 20,000 x 8
for 20 minutes and the precipitate was discarded. The remaining solution was
centrifuged again at 30,000 x g for 30 minutes. This supernatant layer (s-30)
was recentrifuged at 105,000 x g for 2 hours to sediment the ribosomes. The
supernatant solution (S-100) was aspirated and the ribosomes were washed in
standard buffer and centrifugation as before (W-Rib). Fractions 8-30, g-100,
and W-Rib were dialyzed against standard buffer overnight at 5°, and were

stored at -15°.

——
* Supported by a NATO Postdoctoral Research Fellowship

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Vol. 4, No. 6, 1961 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

In most cases fresh $-30 was incubated for 40 minutes at 35° under the
conditions described in Table 1, except that ct valine was present in place
of u-c!4y-valine. After incubation the reaction mixture was dialyzed 10 hours
at 5° against standard buffer which was changed once. The enzyme was stored
at -15°. (Incubated-S-30).

RNA fractions were prepared by three phenol extractions at 24° of fresh
washed ribosomes; 1, 1/2 and 1/2 volumes of H,O-saturated phenol were used
which were concentrated by precipitation with 2 volumes of ethanol and
exhaustively dialyzed against standard buffer minus mercaptoethanol.

In Fig. 1 amino acid incorporation into protein is presented as a function
of time. In the absence of E. coli ribosomal RNA, amino acid incorporation
ceases after 30 minutes. Saturating concentrations of E. coli soluble RNA were
present in every reaction mixture. Increasing the concentration of soluble RNA
3-fold did not increase the amino acid incorporation into protein. However,
when ribosomal RNA was added, a marked increase in amino acid incorporation
occurred. At low concentrations of ribosomal RNA, maximum amino acid incorpor-
ation into protein was proportional to the amount of ribosomal RNA added,
suggesting a stoichiometric rather than catalytic action of ribosomal RNA. In
contrast, soluble RNA has recently been shown to act in a catalytic fashion (3).

In Table 1 are presented the characteristics of cl 4_1-valine incorporation
into protein due to the addition of ribosomal RNA. Amino acid incorporation was
dependent upon the addition of ATP and an ATP-generating system and was com-
pletely inhibited by the addition of RNAase but not DNAase. Heating ribosomal
RNA to 100° for 10 minutes did not destroy its activity. Amino acid incorporation
was inhibited by 0.15 ymoles/ml chloramphenicol and 0.20 pmoles/ml puromycin. The
addition of 20 different L-amino acids markedly stimulated the incorporation of
cl _valine into protein. Therefore, amino acid incorporation due to the addition
of ribosomal RNA had many characteristics expected of protein synthesis.

In all experiments amino acid incorporation was dependent upon the addition

of both ribosomes and 105,000 x g supernatant fluid, indicating that contamination

405
Vol. 4, No. 6, 1961 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

 

1000 }- 4
900,- 7
800)-
700
600
500
400
300
200
100

0.60 mg. RNA

0.30 mg. RNA —

COUNTS / MINUTE

0.12 mg. RNA
Minus RNA -

 

 

 

tL Jt | | | jf jf {| 4
0 10 20 30 40 50 60 70 80 90

MINUTES

Fig. 1 - Dependence of cl4-t-valine incorporation into protein upon
ribosomal RNA. The reaction mixture is presented in Table 1. Each reaction
mixture contained 0.98 mg of E. coli soluble RNA (saturating concentration)
and 4.4 mg of Incubated-S-30 protein.

———_—
of enzyme preparations by intact cells was highly unlikely. Ribosomal RNA had no
effect upon amino acid incorporation into protein in the absence of ribosomes and
the RNA could not be replaced by the addition of equivalent concentrations of poly-
anions such as polyadenylic acid, highly polymerized salmon sperm DNA, or a high
molecular weight polymer of glucose carboxylic acid. Pretreatment of ribosomal
RNA with trypsin did not affect its biological activity. However, treatment of
the ribosomal RNA with RNAase (followed by removal of protein), or alkali, com
pletely destroyed its biologic activity. Thus, all of the activity appeared to
be associated with RNA.

Preliminary attempts at fractionation of the ribosomal RNA have. been per formee

: f
by means of density-gradient centrifugation in sucrose (4). Biological activity °

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Vol. 4, No. 6, 1961 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE 1

Characteristics of cl 4_y-valine Incorporation into Protein

 

 

Expt. No. Counts/minute/mg protein
1 - Ribosomal RNA 42
+ w “" 204
+ " " + 0.15 pmole Chloramphenicol 58
+ " " + 0.20 pmole Puromycin 7
+ " " Zero time 8
2 - Ribosomal RNA 35
+ 1" " 101
+ " "= ATP, PEP, PEP kinase 7
+ " " + 10 pg RNAase 6
+ " "+ 10 pg DNAase 110
+ Boiled Ribosomal RNA . 127
+ Ribosomal RNA, Zero time 8
3 - Ribosomal RNA 34
- 7 " = 20 L-amino acids 21
+ tt “" 9 9g
+ " "= 20 Leamino acids 52

 

The reaction mixtures contained the following in pmole/ml: 100, Tris(hydroxy-
methyl)aminomethane, pH 7.8; 10 Mg acetate; 50 KC1; 6.0 mercaptoethanol;

1.0 ATP; 5.0 phosphoenolpyruvate, K salt; 20 pg phosphoenolpyruvate kinase,
erystalline; 0.05 each of 20 L-amino acids minus valine; 0.03 each of GIP, CTP
and UTP; 0.019 cl4.,-valine (# 70,000 counts); 3.1 mg E. coli ribosomal RNA
where indicated, and 1.0 mg E. coli soluble RNA (saturating concentrations)3
3.2, 3.2 and 1.4 mg of Incubated-S-30 protein were present in Expts. 1, 2, and
3, respectively. In addition 4.4 mg protein of W-Rib were added in Expt. 3.
Total volume was 1.0 ml. Samples were incubated at 35° for 20 minutes, were
deproteinized with 10% trichloracetic acid and the precipitates were washed
and counted by the method of Siekevitz (2).

 

the RNA did not follow absorbancy at 260 mi, instead the activity was concentrated
around a fraction sedimenting about three times as fast as soluble RNA. Investi-
gation of ribosomal RNA preparations with the Spinco Model E ultracentrifuge showed
two major peaks with Soow values of about 23, 16 and a minor one at 4.4, correspond~
ing to those described by Aronson and McCarthy (5). Addition of RNAase, but not
trypsin, destroyed these peaks. It is possible that part or all of the ribosomal
RNA used in our study corresponds to template or messenger RNA. The function of

the ribosomal RNA in our system and its further purification are being studied now.

In summary, a stable, cell-free system has been obtained from E. coli in which

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Vol. 4, No. 6, 1961 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the incorporation of amino acids into protein was dependent on the addition of heat-
stable ribosomal RNA preparations. Soluble RNA could not replace ribosomal RNA
fractions. In addition, the amino acid incorporation required both ribosomes and
105,000 x g supernatant solution. The kinetics of the incorporation suggested
stoichiometric rather than catalytic activity of ribosomal RNA. The ribosomal
RNA-dependent amino acid incorporation also required ATP and an ATP-generating
system, was stimulated by a complete mixture of L-amino acids, and was markedly

inhibited by puromycin, chloramphenicol and RNAase.

References

1. Nirenberg, M. W., and Matthaei, H. (In preparation).

2. Siekevitz, P., J. Biol. Chem., 195, 549 (1952).

3. Hoagland, M. B., and Comly, L. T., Proc. Nat. Acad. Sci., 46, 1554 (1960).
4, Britten, R. J., and Roberts, R. B., Science 131, 32 (1960).

5. Aronson, A. I., and McCarthy, B. J., Biophys. J., 1, 215 (1961).

408