I31       CELL-FREE PROTEIN SYNTHESIS BY MESSENGER RNA      17

[3] Cell-Free Protein Synthesis Directed by
       Messenger RNA

By MARSHALL W. N~RENBERG

Assay Method

PrincipEe. Cell-free extracts of Escherichia coli incorporate C14-amino
acids into protein. Under certain conditions this amino acid incorporation
is dependent on the addition of synthetiF or natura1*~4 messenger RNA,
thus providing a sensitive assay for such RNA fractions. In the absence
of added messenger RNA, amino acid incorporation presumably is
directed by preformed and newly synthesized endogenous messenger
RNA. Preincubation of extracts with DNase prevents new messenger
RNA synthesis and depletes endogenous messenger RNA. C14-Amino
acid incorporation by these extracts is therefore dependent on added
messenger RNA and over a certain range is proportional to the amount
added.

E. coli extracts ("preincubated" S-30 fractions) are incubated with
an ATP-generating system, a C4- amino acid, nineteen C12-amino acids,
and messenger RNA. After incubation, proteins are precipitated and
treated to remove C"-amino acyl transfer RNA and free C14-amino acid.
The Cl" in the washed protein precipitates then is determined.

Reagents

1. Mix I contains: 10.0 ml. of 2M Tris buffer, pH 7.8; 2.0 ml. of
  1.4M magnesium acetate; 5.0 ml. of 21M KCl; 3.0 ml. of 6.66 x
1 M. W. Nirenberg and J. H. Matthaei, Proc. Natl. Acad. Sci. US. 47, 1588 (1961).
`J. H. Matthaei, 0. W. Jones, R. G. Martin, and M. W. Nirenberg, Proc. Natl.
Acad. Sci. VS. 48, 666 (1962).
`J. F. Speyer, P. Lengyel, C. Basilio, and S. Ochoa, Proc. Natl. Acad. Sci. US. 48,
63 (1962).
`A. Tsugita, H. Fraenkel-Comat, M. W. Nirenberg, and J. H. Matthaei, Proc. Natl.
Acad. Sci. US. 48, 846 (1962).


18            ENZYMES OF NUCLEIC ACID METABOLISM       131

1O-2 M ATP, Na salt, and 2.0 X lo-" M GTP, Na salt. Store in
5-ml. aliquots at -20".

2. Phosphoenolpyruvate kinase, crystalline, 10 mg./ml., obtained as
a suspension in (NH,) ,SO, solution (California Biochemical
Corp.).

3. 7.5 X 1O-2 M phosphoenolpyruvate, K or Na salt, crystalline
(California Biochemical Corp.) Store in small aliquots at -20".
The tricyclohexyiammonium salt of phosphoenolpyruvate inhibits
C14-amino acid incorporation and should be converted to the
potassium salt as follows: 5 g. of phosphoenolpyruvate, tricyclo-
hexylammonium salt, are dissolved in minimal quantities of H,O
at 3', and the solution is added to a washed Dowex 50 (H+)
column of approximately 2.5 X 15 cm. Phosphoenolpyruvic acid
is eluted with H,O, and the pH of the eluate is determined rapidly
with pH paper. When the pH of the eluate drops to 2, collection
of the solution is begun, after the pH rises to 4, collection is
discontinued. The phosphoenolpyruvic acid solution is converted
to the potassium salt by the addition of M KOH with vigorous
stirring. The pH of the final solution should be 5.5 to 6.0.
4. 2-mercaptoethanol.
5. C'I-L-Amino acid. 1O-2 M, 4 mc./millimole.
6. Mixture of nineteen C12-L-amino acids, minus the appropriate
C14-amino acid, 2 X l(Y M (each amino acid).
7. Poly U (Miles Chemical Co., Clifton, New Jersey), 30 O.D. units/
ml. at 260 rnp, or other messenger RNA preparation. Poly U
solutions should be stored at -20" in small aliquots. It is im-
portant to note that poly U undergoes a conformational change at
6', so that solutions which have been frozen and thawed may have
lower absorbancies at 260 rnp.

8. "Preincubated" S-30 or S-100 and W-RIB enzyme fractions, de-
scribed below.

Procedure. (a) For ten reaction mixtures, prepare mix II each day as
follows: 250 ~1. of mix I; 1 ~1. of 2-mercaptoethanol; 250 ~1. of phospho-
enolpyruvate, 4 ~1. of phosphoenolpyruvate kinase suspension; 250 ,~l. of
C12-amino acid mixture, and 50 ~1. of Cl'-amino acid solution.

(b) Add components of each fraction mixture in the following se-
quence: SO ~1. of mix II; appropriate amount of H20 to make final
volume 0.25 ml. ; 5 to 20 ~1. of poly U solution (15 to 20 pg. of poly U or
100 pg. of natural messenger RNA usually saturates a reaction mixture) ;
and E. coli extracts (0.5 to 2 mg. of protein). Reaction mixtures are
incubated at 37" for 15 minutes. C14-Amino acid incorporation should be


I31       CELL-FREE PROTEIN SYNTHESIS BY MESSENGER RNA      19

dependent on the addition of messenger RNA, and, when limiting
amounts of messenger RNA are added (see Remarks), incorporation
should be proportional to the amount of messenger RNA added.

(c) Protein is precipitated at the end of incubation by the addition
of 3.0 ml. of 10% trichloroacetic acid at 3", and precipitates are washed
by the method of Siekevitz.5

For exploratory work and preparation of extracts, the following fast
procedure6 for washing protein precipitate8 may be used for all Cl"-amino
acids incorporations except P4-tryptophan. Reaction mixtures depro-
teinized by the addition of 10% trichloroacetic acid are placed in a
water bath at 90" to 95" for 20 minutes to hydrolyze amino acyl transfer
RNA. Tubes then are chilled in ice for 30 minutes. Protein precipitate8
are dispersed by stirring or by vigorous agitation, and each suspension
is filtered under suction through a Millipore filter (HA Millipore filter,
25 mm. in diameter, 0.45-p pore size, held in an appropriate Millipore
filter funnel, Millipore Co., Bedford, Massachusetts). Each precipitate
then is washed rapidly with five 5-ml. aliquots of cold 5% trichloroacetic
acid. If the radioactivity of the washed protein precipitate is to be
counted in a thin-window gas-flow B-scaler, the Millipore filter is glued
with rubber cement to a disposable planchette, dried for 5 to 10 minutes
under an infrared lamp, and counted. Curled Millipore filter edges should
be reglued, for they will tear window8 of gas-flow counters.

Growth of E. coli

Escher&u coli W-3100 (other strains may be used) are grown at
temperatures between 25" and 37" (30" is advantageous) in a medium
containing 8 g. of nutrient broth (Difco Co., Detroit, Michigan) and
5 g. of glucose per liter. A 60% solution of glucose is autoclaved and is
added to autoclaved nutrient broth solution after both have cooled. A
typical preparation consists of five 19-l. carboys, each containing 15 1. of
nutrient broth-glucose solution and equipped for aeration. About 1 g. of
Antifoam A (Dow-Corning Corp.) is suspended with stirring in approxi-
mately 50 ml. of H,O, and the suspension is centrifuged at 3000 X g for
10 minutes to remove undissolved lumps. The supernatant solution of
antifoam is autoclaved, and approximately 1 to 2 ml. are added to each
carboy before inoculation with bacteria. Additional antifoam may be
needed to prevent foaming during growth of bacteria; however, minimal
amount8 should be used. One liter of nutrient broth-glucose solution in a
6-1. Ehrlenmeyer %ask is inoculated with E. coli grown on the surface

`P. Siekevitz, J. Biot. Chem. 195, 519 (19%).
`M. W. Nrenberg, unpublished data.


20           ENZYMES OF NUCLEIC ACID METABOLISM       [31

of an agar slant, and the flask is aerated by shaking at 25" for 8 to 12
hours. While the culture is still in the logarithmic growth phase, 200-ml.
aliquots are added to each carboy. The carboys are vigorously aerated
for 1.5 to 6 hours, depending on the temperature of the medium and the
degree of aeration. At 30", 2.5 to 3.0 hours of growth will sufEce. It &
important to follow cell growth carefully so that aeration may be stqped
at the proper time. Aliquots of cultures should be removed at intervals
so that turbidities may be determined spectrophotometrically. Aeration
should be stopped when a reading of 25 to 35 Klett spectrophotometer
units at 520 rnp is obtained, compared to a sterile nutrient broth-glucose
solution set at zero. The carboys then are chilled in ice water, and the
cells are harvested by means of a refrigerated Sharpless centrifuge. A
yield of 0.5 to 0.75 g. of packed cells, wet weight, per liter of medium is
optimum. When more cells are obtained, the extracts often are less active.
The packed cells are washed by rapid suspension in 3 vol. (w/v) of
0.01 M Tris, pH 7.8, 0.014 M magnesium acetate, and 0.06 M potassium
chloride at 3", and are centrifuged at 15,000 x g in preweighed tubes
for 15 minutes at 3'. The supernatant solutions are decanted, and the
pellets are drained. Centrifuge tubes again are weighed to obtain wet
weights of packed cells. Washed, packed cells may be frozen quickly
and stored for several months at -20" if desired.

Reparation of Extracts

Step 1. All operations, unless specified, are carried out in a cold room
or at 3". Cells may be disrupted either by grinding with alumina in a
mortar, or with a French press (American Instrument Co.). If the
alumina grinding method is to be used, about 30 g. of fresh or thawed
packed cells are transferred to a large prechilled, unglazed porcelain
mortar (20 cm. in diameter), and 30 g. of alumina A-301 (Aluminum
Corporation of America) are added. The cells are ground vigorously
with a pestle into a thick paste; cracking, popping noises should be
produced during grinding. As the cells break, the paste becomes more
fluid, and an additional 30 g. of alumina are slowly added to keep the
paste thick and to maintain the popping noise. After about 15 minutes
of grinding, 60 ml. of "standard buffer" containing 0.01 M Tris, pH 7.8,
0.014 M magnesium acetate, 0.06M potassium chloride, and 0.006M
2-mercaptoethanol freshly prepared are added to the mortar, and the
paste is suspended evenly by gently stirring. Alumina, intact cells, and
debris are removed by centrifugation at 20,000 X g for 20 minutes. The
supernatant fluid is decanted, and the pellet is discarded. Cells to be
broken in a French press should be evenly suspended in 1.5 to 2.0 vol.
of "standard buffer" (w/v) and disrupted with 18,000 p.s.i. in a pre-


131       CELL-FREE PROTEIN SYNTHESIS BY MESSENGER RNA      21

chilled cylinder. Additional 2-mercaptoethanol (6 micromoles/ml. of
extract) is added after the cells have been broken.

Step b. Extracts disrupted in either manner are treated as follows:
Two micrograms of pancreatic DNase (twice crystallized, Worthington,
Biochemical Co.) are added with gentle mixing to each milliliter of
E. coli extract. (Caution: Some commercial DNase preparations are
contaminated with pancreatic RNase, which strongly inhibits C14-amino
acid incorporation into protein.) Before the DNase is added, the extract
obtained by alumina grinding should be noticeably viscous and should
contain some gelatinous clots. Although the extract is maintained at 3",
5 minutes after addition of the DNase the viscosity and clots disappear.
The extract is centrifuged at 30,000 )( g for 30 minutes at 3". The
supernatant solution is removed by aspiration to within 1 cm. of the
pellet and again is centrifuged at 30,000 X g for 30 minutes. The upper
four-fifths of the supernatant solution is removed by aspiration, and
henceforth will be referred to as the S-30 fraction.

At this stage the activities of 25 to 150-~1. aliquots of the S-30 frac-
tion should be assayed rapidly to determine how much C14-phenylalanine
can be incorporated into protein in the presence and in the absence of
poly U. One milligram of S-30 protein added to a 0.25-ml. reaction mix-
ture containing 15 to 20 pg. of poly U should incorporate 1 to 20 milli-
micromoles of C"-phenylalanine into protein in 15 minutes at 37'. If,
in the absence of poly U, 1 mg. of S-30 protein directs more than 0.1
millimicromole of C'4-phenylalanine into protein, the "preincubation"
procedure described in step 3 should be followed. (Step 3 usually is neces-
sary.) If less than 0.1 millimicromole of Cl*-phenylalanine is incorpo-
rated in the absence of poly U, the S-30 fraction may be dialyzed for 8
hours as described in step 3.

For routine messenger RNA assays and many amino acid incorpora-
tion studies, ribosomes need not be separated from the S-30 fraction. If
the S-30 preparation at this stage can be used for assays, the dialyzed
extract should be divided into I- to 3-ml. aliquots (one tube of extract
per experiment), frozen quickly, and stored until needed.

Step 3. "Preincubation'." S-30 extracts carried through this step are
almost completely dependent on added messenger RNA.

The components of a reaction mixture appropriate for "preincuba-
tion" of 100 ml. of S-30 are: 10 ml. of M Tris, pH 7.8; 2.0 ml. of 0.14M
magnesium acetate; 4.0 ml. of 0.02M ATP (neutralized); 12 ml. of
7.5 X lo-* M phosphoenolpyruvate, K or Na salt; 1 mg. of pyruvate
kinase, crystalline; 0.04 ml. of 2-mercaptoethanol; and 1 micromole each
of twenty L-amino acids. After addition of 100 ml. of S-30 fraction, the
reaction mixture is incubated at 37" for 80 minutes. The reaction mix-


22          ENZYMES OF NUCLEIC ACID METABOLISM       [31

ture then is cooled to 3" and dialyzed against 120 vol. of "standard
buffer" at 3" for 8 hours. The dialyzing medium should be changed once.
In most cases the `Lprcincubatcd" S-30 fraction can be used after dialysis
without further preparation to assay messenger RNA. The extract is
divided into l- to 3-ml. aliquots and frozen quickly for storage.

One milligram of dialyzed "preincubated" S-30 protein added to a
0.25-ml. reaction mixture containing 15 to 20 pg. of poly U (see Remarks)
should incorporate 1 to 40 millimicromoles of C14-phenylalanine into pro-
tein during 15 minutes of incubation at 37'.

Step 4. If washed ribosomes and supernatant solution are required,
the dialyzed S-30 extract is centrifuged at 105,000 )( g for 120 minutes
in a Spinco Model L preparative ultracentrifuge at 3". The upper four-
fifths of the supernatant solution is aspirated (S-100 fraction). The
lower fifth of the supernatant solution is decanted and discarded. Ribo-
somal pellets are suspended in the initial volume of "standard buffer" by
gentle homogenization (four to five passes) in a Potter-Elvejehm homog-
enizer. The ribosomal suspension is centrifuged again at 105,000 X g for
2 hours, and the supernatant solution is decanted and discarded. The
ribosomal pellets are suspended, as described previously, in one-fourth
the original volume of "standard buffer," and the suspension is centri-
fuged at 10,000 X g for 5 minutes to remove aggregates. The washed
ribosomal fraction will be designated W-RIB. W-RIB and S-100 frac-
tions are divided into small aliquots, frozen quickly, and stored until
needed.

Remarks

Stability of E. coli Extracts. The extracts appear to retain more
activity if they are frozen rapidly in either dry ice-acetone mixture or
in liquid N2 than if they are frozen slowly at -20". Little loss in activity
of S-30 fractions can be detected after storage in liquid N, refrigerators
(Linde Corp.) for 6 months and longer. S-30 fractions stored at -20"
lose less than 5% activity per week. S-100 and W-RIB preparations also
retain more activity when stored under liquid N, than when stored at
-2OO.

Some amino acid-activating enzymes may be more labile than others;
thus, S-30 and S-100 fractions are not frozen and thawed more than once.
However, W-RIB fractions may be frozen and thawed several times
without undue loss of activity.

Template Activities of Poly U Preparations. Optimal amounts of
poly U or natural messenger RNA required for reaction mixtures may
depend on the nuclease content of E. coli extracts.* Another variable
factor related to the template activity of poly U is the average molecular


I34                    RNA POLYMERASE                23

weight of each poly U preparation. Poly U molecules composed of less
than one hundred uridylic acid residues are almost completely inactive.2
Therefore, poor C14-phenylalanine incorporations need not be ascribed
always to inactive E. coed extracts.

Most poly U preparations contain traces of nucleases; to prevent
degradation, solutions may be stored in small aliquots at -20' and kept
at 3' when thawed.