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Sensitization of bacterial ribosomes for in vitro amino acid incorporation
BIOCHEMICAL
Vol. 12, No. 4,1963
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
SENSITIZATION OF BAcflERIAL RIBOSOMESFORINVITRO -AMINOACID INCORPQRATIUN* Seymour Lederberg,
Boris Rotman, and Victoria
Lederberg
Department of Biology, Brown University, Providence, Rhode Island, and Syntex Institute for Molecular Biology, Palo Alto, Cslifornia.
ReceivedJunelS,
1963
The addition of bacterial
of the supernatant
extracts
somes, transfer
acids into
acid-insoluble
speed supernatant incorporation
or native
supply stimulates
and characterization
ability
to suspensions containing
RNA, synthetic
energy generating
of a high speed centrifugation
to respond.
We present
factors
ribosomes without
ribosomes by sedimenting
them in a high ionic
whose density
than that
Methods:
Cells
in a glycerol prepared acid,
medium were employed (5).
A linear
coli
tubes by mixing
impairing
preparing strength
strain
sensitized solution
Ribosomal extracts
were
(O.O,l g magnesium acetate,
of CsCl was prepared
CsCl solutions
their
grown
by the method of Tissi&es,
gradient
the background
K-12,
tris(hydroxymethyl)aminomethane,
pII 7.4)
in the high
of the average protein.
of wild type Escherichia
in TSM buffer
0.014
ethanol;
is greater
The isolation
present
by reducing
a method for
and an of amino
(1, 2, 3, 4).
of the stimulating would be facilitated
ribs-
polynucleotides,
the incorporation
polypeptides
of unstimulated
bacterisl
0.004 g succinic
0.006 E P-mercaptoet.
al.
in plastic
(1). centrifuge
of 1.3 g/cm3 and 1.6 g/cm3 made in
*This investigation was supported by grants from the National Institutes of Health, U.S. Public Health Service, and from the National Science Foundation. Contribution No. 7 from the Syntex Institute for Molecular Biology. 324
BIOCHEMICAL
Vol. 12, No. 4,1963
TSM buffer
containing
solutions
1.3
g/cm3
swinging bucket rotor
on top of the gradient.
at 35,000 rpm for
The
2 to 6 hours in a
SW139 in a Spinco L centrifuge.
by puncturing
drop fractions,
were added simulta-
preparations to make a solution of ++ and 0.04 g in Mg . Volumes of 0.1 to 0.4 ml
was centrifuged
were recovered
Concentrated
ribosomal
of these ribosomes were layered preparation
RESEARCH COMMJNICATIONS
0.03 E magnesium acetate.
of CsCl and magnesium acetate
neously to chilled density
AND BIOPHYSICAL
or for
Samples
the bottom of the tube and collecting
prolonged
centrifugations,
by slicing
the
tube and removing the pellet. content
Ribosomsl
260 w.
Fractions
in TSI buffer, pellet,
for
containing
ribosomes were pooled,
and centrifuged
now completely
resuspended,
was determined by the optical
incorporation
and transparent,
to insure
studies.
at
diluted
at 39,000 rpm for 120 min.
colorless
recentrifuged
density
The
was
removal of CsCl, and used
Ribosomes washed an equal number of
times in TSM, but not exposed to the salt
density
gradient,
were
also employed. Transfer Ofengand, et.
RNA was prepared sl.
(6).
Dr. W. E. Razzell, The supernatsnt original
from -E. coli
Polyuridylic
of the first
cell-free
extract
to remove traces
against
TSM buffer.
100
Imoles
was centrifuged of ribosomes,
up to 5 mg ribosomes,
pmole
then dislysed
mixture
tris,
10
pmoles
ATP, 0.03
pmoles
of the
at 45,000 rpm for 18 hours
in 1 ail final
up to 0.2 ml dialysed
RNA, 0.45 0.D.260mcL units
6 pmoles 20mercaptoethanol, 1
from
39,000 rpm centrifugation
The complete incorporation
1.1 mg transfer
acid was a gift
or was purchased from Miles Laboratories.
90 tin.
contained
by the method of
magnesium
acetate,
volume supernatant,
of polyuridylic 60
pmoles
acid, KCl,
5 pmoles phosphoenolpyruvate, each of GTP, CTP, and UTP, 20 pg 325
Voi.12,No.4,1963
BlOCHEMlCAl.
pyruvate slanine
kinase,
and 9 mmoles uniformly
(21 uc/crmole);
Mixtures
were incubated
in 1s
at 37' C,
labeled
after
washed, dried,
pore membrane filters,
with
was stopped
centrifugation
was
in 5% TCA, and heated for
Samples were chilled,
Chicago flow counter
CIQ-phenyl-
The reaction
The precipitate
NaOH, reprecipitated
15 min. at 90' C.
RESEARCH COMMUNICATIONS
pH 7..8.
with one ml of 15% TCA. dispersed
AND BIOPHYSICAL
filtered
through
millin
and counted in a Nuclear
an efficiency
of 2&30 percent.
A
background of 16 cpm and a zero time value of 3 to 7 cpm have been subtracted
from the data presented.
TAPE1 Incorporation of ClQ-phenylslanine into ribosomes prepared by washing in buffer CsCl gradient.
Ribosomes
Cradienttreated
(mg)
Bufferwashed
acid-insoluhle product by or by centrifu&ation in a
Supernatant WI
Preparation
Acid-insoluble Product (counts/min) 1
5
(1
5
1759
0.15 5
65
5 5*
0.15
283
0.15
55
Prenaration
2 5
0.22
0.15
0.22
139
0.22 0.22
*
Polyuridylic of all l-amino
1028
0.15
316
acid was omitted from this sample, and 0.5 wmoles acids other than phenylslanine was added. 326
Vol. 12, No. 4,1963
Results:
BIOCHEMICAL
The results
of amino acids into
in Table 1 indicate protein
essentially
nil
is markedly
stimulated
reduction treated natant
AND BIOPHYSICAL
that
the incorporation
by gradient-treated
ribosomes is
in the absence of the supernatant by its
of background incorporation ribosomes is effected
of this
hypothesis,
bound enzymes (5, 7),
this
to carry for
supernatant
relative
and similar
(Table 1).
to ribosomes,
of super-
adhere to washed ribosomes.
we note that
in studies
This suggests that
results
In
of ribosomal-
methods of preparing proteins.
in their We find
absolute
ability
sn optimum ratio
above which an increase
amount of supernatant
(Table 2).
the
by the removal of traces
show an increase
out incorporation
and
by unsupplemented gradient-
ribosomes have been used to remove contaminant Some preparations
fraction,
Presumably,
presence.
components which otherwise
support
RESEARCH COMMUNICATIONS
in the
in decreased incorporation
salt-gradient
treatment
removes an
TABLE: 2 Effect of supernatsnt fraction on incorporation of C14-phenylalanine into acid-insoluble product by gradient-treated ribosomes.
Gradient-treated ribosomes (mg)
Supernatant WQ
Acid-insoluble product (counts/min) 1
2
*
1
0.05
1260
1
0.1
ll70
1
0.2
310
0.2
41
2
0.2
2*
0.2
Polyuridylic of all l-amino
2665 66
acid was omitted from this sample, and 0.5 pmoles acids other than phenylalanine was added. 327
SlOCHEMlCAl
Vol. 12, No. 4, 1963
inhibitor
or nuclease,
ribosomes, acid.
additional reintroduce
or releases
thus revealing
Restoring factors
AND BIOPHYSICAL
inactive
new attachment
the supernatant necessary for
the antagonizing
RESEARCH COMMUNICATIONS
fraction
messenger-RNA from sites
for
added polyuridylic
not only provides
incorporation,
but may
the
also
agent. References
1.
2. 3. 4. 5. 6. 7.
Tissi&es, A., Schlessinger, D., and Gros, F., Proc. Nat. Acad. Sci., U.S., 46, 1450 (1960). Lamborg, M. R. and Zsmecnik, P. C., Biochim. Biophys. Acta, 42, 206. 0960). Nathsns, D. and Lipmann, F., Proc. Nat. Acad. Sci., U.S., 47, 497 0961). Nirenberg, M. W, and Matthei, J. H., Proc. Nat. Acad. Sci., U.S., 47, 1588 (1961). Lederberg, S., Rotman, B,, and Lederberg, V., (in preparation). Ofengand, E. J., Dieckmann, M., and Berg, P., J. Biol. Chem., 236, 1741 (1961). Spahr, P. F. and Hollingworth, B. R., J. Biol. Chem., 236, 823 (1961).
328
Vol. 12, No. 4,1963
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
SENSITIZATION OF BAcflERIAL RIBOSOMESFORINVITRO -AMINOACID INCORPQRATIUN* Seymour Lederberg,
Boris Rotman, and Victoria
Lederberg
Department of Biology, Brown University, Providence, Rhode Island, and Syntex Institute for Molecular Biology, Palo Alto, Cslifornia.
ReceivedJunelS,
1963
The addition of bacterial
of the supernatant
extracts
somes, transfer
acids into
acid-insoluble
speed supernatant incorporation
or native
supply stimulates
and characterization
ability
to suspensions containing
RNA, synthetic
energy generating
of a high speed centrifugation
to respond.
We present
factors
ribosomes without
ribosomes by sedimenting
them in a high ionic
whose density
than that
Methods:
Cells
in a glycerol prepared acid,
medium were employed (5).
A linear
coli
tubes by mixing
impairing
preparing strength
strain
sensitized solution
Ribosomal extracts
were
(O.O,l g magnesium acetate,
of CsCl was prepared
CsCl solutions
their
grown
by the method of Tissi&es,
gradient
the background
K-12,
tris(hydroxymethyl)aminomethane,
pII 7.4)
in the high
of the average protein.
of wild type Escherichia
in TSM buffer
0.014
ethanol;
is greater
The isolation
present
by reducing
a method for
and an of amino
(1, 2, 3, 4).
of the stimulating would be facilitated
ribs-
polynucleotides,
the incorporation
polypeptides
of unstimulated
bacterisl
0.004 g succinic
0.006 E P-mercaptoet.
al.
in plastic
(1). centrifuge
of 1.3 g/cm3 and 1.6 g/cm3 made in
*This investigation was supported by grants from the National Institutes of Health, U.S. Public Health Service, and from the National Science Foundation. Contribution No. 7 from the Syntex Institute for Molecular Biology. 324
BIOCHEMICAL
Vol. 12, No. 4,1963
TSM buffer
containing
solutions
1.3
g/cm3
swinging bucket rotor
on top of the gradient.
at 35,000 rpm for
The
2 to 6 hours in a
SW139 in a Spinco L centrifuge.
by puncturing
drop fractions,
were added simulta-
preparations to make a solution of ++ and 0.04 g in Mg . Volumes of 0.1 to 0.4 ml
was centrifuged
were recovered
Concentrated
ribosomal
of these ribosomes were layered preparation
RESEARCH COMMJNICATIONS
0.03 E magnesium acetate.
of CsCl and magnesium acetate
neously to chilled density
AND BIOPHYSICAL
or for
Samples
the bottom of the tube and collecting
prolonged
centrifugations,
by slicing
the
tube and removing the pellet. content
Ribosomsl
260 w.
Fractions
in TSI buffer, pellet,
for
containing
ribosomes were pooled,
and centrifuged
now completely
resuspended,
was determined by the optical
incorporation
and transparent,
to insure
studies.
at
diluted
at 39,000 rpm for 120 min.
colorless
recentrifuged
density
The
was
removal of CsCl, and used
Ribosomes washed an equal number of
times in TSM, but not exposed to the salt
density
gradient,
were
also employed. Transfer Ofengand, et.
RNA was prepared sl.
(6).
Dr. W. E. Razzell, The supernatsnt original
from -E. coli
Polyuridylic
of the first
cell-free
extract
to remove traces
against
TSM buffer.
100
Imoles
was centrifuged of ribosomes,
up to 5 mg ribosomes,
pmole
then dislysed
mixture
tris,
10
pmoles
ATP, 0.03
pmoles
of the
at 45,000 rpm for 18 hours
in 1 ail final
up to 0.2 ml dialysed
RNA, 0.45 0.D.260mcL units
6 pmoles 20mercaptoethanol, 1
from
39,000 rpm centrifugation
The complete incorporation
1.1 mg transfer
acid was a gift
or was purchased from Miles Laboratories.
90 tin.
contained
by the method of
magnesium
acetate,
volume supernatant,
of polyuridylic 60
pmoles
acid, KCl,
5 pmoles phosphoenolpyruvate, each of GTP, CTP, and UTP, 20 pg 325
Voi.12,No.4,1963
BlOCHEMlCAl.
pyruvate slanine
kinase,
and 9 mmoles uniformly
(21 uc/crmole);
Mixtures
were incubated
in 1s
at 37' C,
labeled
after
washed, dried,
pore membrane filters,
with
was stopped
centrifugation
was
in 5% TCA, and heated for
Samples were chilled,
Chicago flow counter
CIQ-phenyl-
The reaction
The precipitate
NaOH, reprecipitated
15 min. at 90' C.
RESEARCH COMMUNICATIONS
pH 7..8.
with one ml of 15% TCA. dispersed
AND BIOPHYSICAL
filtered
through
millin
and counted in a Nuclear
an efficiency
of 2&30 percent.
A
background of 16 cpm and a zero time value of 3 to 7 cpm have been subtracted
from the data presented.
TAPE1 Incorporation of ClQ-phenylslanine into ribosomes prepared by washing in buffer CsCl gradient.
Ribosomes
Cradienttreated
(mg)
Bufferwashed
acid-insoluhle product by or by centrifu&ation in a
Supernatant WI
Preparation
Acid-insoluble Product (counts/min) 1
5
(1
5
1759
0.15 5
65
5 5*
0.15
283
0.15
55
Prenaration
2 5
0.22
0.15
0.22
139
0.22 0.22
*
Polyuridylic of all l-amino
1028
0.15
316
acid was omitted from this sample, and 0.5 wmoles acids other than phenylslanine was added. 326
Vol. 12, No. 4,1963
Results:
BIOCHEMICAL
The results
of amino acids into
in Table 1 indicate protein
essentially
nil
is markedly
stimulated
reduction treated natant
AND BIOPHYSICAL
that
the incorporation
by gradient-treated
ribosomes is
in the absence of the supernatant by its
of background incorporation ribosomes is effected
of this
hypothesis,
bound enzymes (5, 7),
this
to carry for
supernatant
relative
and similar
(Table 1).
to ribosomes,
of super-
adhere to washed ribosomes.
we note that
in studies
This suggests that
results
In
of ribosomal-
methods of preparing proteins.
in their We find
absolute
ability
sn optimum ratio
above which an increase
amount of supernatant
(Table 2).
the
by the removal of traces
show an increase
out incorporation
and
by unsupplemented gradient-
ribosomes have been used to remove contaminant Some preparations
fraction,
Presumably,
presence.
components which otherwise
support
RESEARCH COMMUNICATIONS
in the
in decreased incorporation
salt-gradient
treatment
removes an
TABLE: 2 Effect of supernatsnt fraction on incorporation of C14-phenylalanine into acid-insoluble product by gradient-treated ribosomes.
Gradient-treated ribosomes (mg)
Supernatant WQ
Acid-insoluble product (counts/min) 1
2
*
1
0.05
1260
1
0.1
ll70
1
0.2
310
0.2
41
2
0.2
2*
0.2
Polyuridylic of all l-amino
2665 66
acid was omitted from this sample, and 0.5 pmoles acids other than phenylalanine was added. 327
SlOCHEMlCAl
Vol. 12, No. 4, 1963
inhibitor
or nuclease,
ribosomes, acid.
additional reintroduce
or releases
thus revealing
Restoring factors
AND BIOPHYSICAL
inactive
new attachment
the supernatant necessary for
the antagonizing
RESEARCH COMMUNICATIONS
fraction
messenger-RNA from sites
for
added polyuridylic
not only provides
incorporation,
but may
the
also
agent. References
1.
2. 3. 4. 5. 6. 7.
Tissi&es, A., Schlessinger, D., and Gros, F., Proc. Nat. Acad. Sci., U.S., 46, 1450 (1960). Lamborg, M. R. and Zsmecnik, P. C., Biochim. Biophys. Acta, 42, 206. 0960). Nathsns, D. and Lipmann, F., Proc. Nat. Acad. Sci., U.S., 47, 497 0961). Nirenberg, M. W, and Matthei, J. H., Proc. Nat. Acad. Sci., U.S., 47, 1588 (1961). Lederberg, S., Rotman, B,, and Lederberg, V., (in preparation). Ofengand, E. J., Dieckmann, M., and Berg, P., J. Biol. Chem., 236, 1741 (1961). Spahr, P. F. and Hollingworth, B. R., J. Biol. Chem., 236, 823 (1961).
328