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An effect of pactamycin on the initiation of protein synthesis in reticulocytes
Vol. 41,No.
BIOCHEMICAL
1, 1970
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
AN EFFECT OF PACTAMYCIN ON THE INITIATION
OF
PROTEIN SYNTHESIS IN RETICULOCYTES* John S. Macdonald Department.of Beth Israel Received
Medicine, Hospital,
and Irving
H. Goldberg
Harvard Medical School and the Boston, Massachusetts 02215
July 31, 1970
Summary: Low levels of pactamycin (about 10m6 M) stop polypeptide synthesis by intactreticulocytes and their lysates after a lag of about two minutes. This effect which is similar to that of poly A, an inhibitor of polypeptide chain is not additive to that of poly A. Cells which have been preinitiation, treated with NaF and must form new polypeptide chains are more sensitive to pactamycin than cells which are able to complete pre-existing chains. High levels of pactamycin (&1O-5 M) inhibit elongation in addition to initiation. ‘H-pactamycin binds to 80s ribosomes and the 40s subunits at 0’. Monosomes very effectively, produced by NaF treatment of reticulocytes bind 5H-pactamycin whereas those produced by RNase treatment of polyribosomes do not. Pactamycin whole
cells
shown
that
(PM) is
and extracts PM binds
structure
a potent
of the
phenylalanyl-tRNA,
antibiotic
of procaryotic
to the
initiator
and eucaryotic
complex
N-formylmethionyl-tRNA,
(with
of polypeptide
synthesis
of inhibition
does not effect
interfer
is with
also
data,
the conversion
coupled of rabbit
of soluble,
greater
newly
the
N-aaetylresulting
synthesis
in
itself,
its
PM inhibition is greater has started;
at low Mg++ concentrations.’
bond formation
the
PM, however,
as judged
by its
lack
the finding
reticulocyte synthesized
of Colombo,
polyribosomes protein,
al -et 2)
’ that
to 80s ribosomes
suggest
that
this
PM induces with
antibiotic
* This work U.S. Public
of
2,4,5,7
reaction. with
and affects
Furthermore,
polypeptide
in
It has been
by N-acetylphenylalanyl-tRNA before
peptide
on the puromycin These
release
initiated
is present
of --E. coli
as “initiator”-tRNA),
at low Mg++ concentrations.5-7
synthesis
cells.l-5
subunit
disassociation
extent
of protein
30s ribosomal
or polylysyl-tRNA
when the antibiotic
inhibitor
was supported by grants from the National Institutes Health Service (GM 12573) and the American Cancer
of Health, Society.
the may
Vol. 41,No.
affect
BIOCHEMICAL
1, 1970
the initiation
of polypeptide
experiments (M. Stewart,
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
synthesis.
I. Yanowitz and I.H.
This view finds support in Goldberg, in preparation),
where low levels of PM (about 10m6M, dependent on the number of ribosomes) rapidly
and almost quantitatively
in lysates from rabbit
produce single ribosomes from polyribosomes
reticulocytes
with the release of completed hemoglobin
chains. Wenow show that low concentrations initiation inhibit
in reticulocytes elongation.
and their
of PMmainly affect
lysates,
polypeptide
chain
but that higher concentrations
also
Furthermore, the ribosomal binding site for PM (at O" and
10-7 M PM) is absent in ribosomes bearing a fragment of mRNA,such as are produced by RNasetreatment of polyribosomes. Materials
and Methods:
and cell-free
The preparation
and incubation of rabbit
lysates were as described by Colombo, et al.,'
lysate system the incorporation acid-precipitable
of ( l4C) valine
reticulocytes
except that in the
into hot trichloroacetic
protein was assayed by the filter
paper disc method described
by Bollum8.
The binding of (3H) PM, prepared as previously
reticulocyte
ribosomes was measured on 15-30% sucrose density gradients contain-
ing 0.01 M Tris,
pH 7.4, 0.01
in the Spinco rotor collected
SW41
KCl, and 0.0015 M MgClz.
analysis as previously
Results and Discussion:
synthesis by reticulocyte
which the particular the incorporation
lysate
with the initiation
varies with the degree to
depends on the formation of new chains for
of labelled amino acids.
Figure 1 shows that in the presence
proceeds at control
rates for almost two
minutes, at which time there is an abrupt cessation of further also shows that 10s6 PM gives essentially
inhibition
as does poly A.
were
absorbance recorder
lysates by interfering
of poly A the synthesis of polypeptide
figure
centrifugation
have shown that poly A inhibits
The extent of inhibition system
to
described.6
Hardesty, et al., '*lo
of new hemoglobin chains.
After
41,000 RPMfor 90 min. at 4O, fractions
from the bottom of the tube through a Gilford
for radioactivity
polypeptide
at
M
reported,6
the same level and kinetics
Furthermore, when poly A and 10-6
2
synthesis.
PM
This of
are both present,
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 41, No. 1, 1970
SPARS
0
2
4
6
8
+ POLY A
10
protein synthesis and poly A on cell-free Figure 1. Effect of antibiotics The incorporation of (14C) valine (35 uc/umole ) into protein by reticulocy t e The volume of the reaction lysates was followed as previously described.l . mixture was 500 ul; 75 1.11was removed for assay at each _. of . ^the indicated times. Poly A (100 ug/ml), PM and sparsomycin were added tram tne start ot the incubation, as indicated
the pattern additive
and extent of inhibition
effect
is that of either one alone; there is no
suggesting that both agents are exerting
the sameoverall
step in protein
maximal actions at
synthesis.
During the two minute lag in onset of the inhibitory
action of PM (and
poly A) nascent peptide chains are presumably being completed and released fron the ribosome. continuously,
In reticulocytes
or lysates where new chains are being formed
the lag in onset of the PM (10-6 M) inhibition
although the final
extent of inhibition
increases as the number of rounds of
synthesis of chains increases in the control. the inhibitory
effect
is always found,
Since PMbinds to the ribosome,
depends on both the PMand ribosome concentrations. 3
Vol.41,No.
BIOCHEMICAL
1, 1970
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Increasing the number of ribosomes but not the amount of soluble protein decreases the PM effect A very different
(Cohen and Goldberg, unpublished results). result
is found with sparsomycin, which is an inhibitor
of peptide bond formation.ll
At the low level of sparsomycin (6 x 10-8 M)
used in Figure 1, the rate of protein start
and this effect
is additive
synthesis is slowed down from the very
with that due to poly A.
(Figure 2A) that at higher concentrations protein biotic
synthesis is also inhibited, polypeptide
rate of
suggesting that at high levels of anti-
to this inhibition.
found at high PM concentrations location
of PM (10-S M), the initial
chain elongation is also affected.
does not contribute
It should be noted
The inhibition
probably results
since peptide bond formation
As expected, poly A of chain elongation
from an effect
(the puromycin reaction)
on transremains intact
(I. Yanowitz and I.H. Goldberg, unpublished data).
A. 3000
CONTROL
8.
Na F
-
2500
5ooc 0
2
4
6
8
10
of NaF-treated and untreated reticulocytes to PM. Figure 2. Sensitivity Reticulocytes were incubated as previously described1 without (A) or with (B) 0.01 M NaF for 30 min. at 37O. After addition of equal volumes of cold saline, the cells were washed four times with the samebuffer prior to reincubation under standard conditions (0.8 nc of 14C-amino acid mix (1.5 mc/mg) per reaction)l in the presence or absence of PM. At the indicated times 100 1.11aliquots were removed for the analysis of 14C incorporation into pretein. 4
Vol. 41, No. 1, 1970
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Figure 2 also shows that the pretreatment
of reticulocytes
with NaF, which
converts most of the polyribosomes to single ribosomes,12 increases the sensitivity
of polypeptide
synthesis is almost entirely chains.
synthesis to PM. In this system, polypeptide dependent on the initiation
Similar results have been found with protein
prepared from NaF treated cells
(M. Stewart,
of new hemoglobin synthesis in lysates
I. Yanowitz and I.H.
Goldberg,
unpublished results). In a system (polyphenylalanine physiological affects
synthesis) that is not dependent on the
mechanismfor the initiation
the overall
of protein
synthesis,
PM (10e5 M)
synthesis only to a small degree and, as with sparsomycin,
acts mainly by decreasing the initial
M/W
rate of synthesis (Figure 3).
TES
Figure 3. Effect of PM on poly U-dependent polyphenylalanine synthesis. S-100 and ribosomal fractions were obtained by centrifugation of a 1:l reticulocyte lysate at 105,OOOxgfor 2 hours. NH4Cl washed ribosomes were prepared as described by Felicetti and Lipmann.13 Polyphenylalanine synthesis was followed by incubating the following in a volume of 200 1.11: 40 ug E. coli (14C) phe-tRNA (330 cpm/ug), 10 umoles Tris, pH 7.4, 16 umoles KCl, 0.8or0.8 umoles GTP, 2 units (A260mu) 1.6 umoles MgC12, 3.2 umoles dithiothreitol, ribosomes, 40 ug poly U, and 30 ul S-100. At the indicated times 30 ul aliquots were removed for assay on filter paper discs.8 PM (lo-5 M) was added as indicated. In the absence of poly U, 200 cpm were incorporated at 15 min. 5
Vol. 41,No.
BIOCHEMICAL
1, 1970
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
PM binds at O" to the 80s ribosome and the 40s ribosomal subunit of the reticulocyte,
but not the polyribosome (Figure 4).
The specific
activity
of
PMbound to 40s subunits is much higher than that bound to 80s ribosomes,
200
-
ii’iI 020 ?5-J ’\:I :? -I 0.10
\
d
I
5
IO
15
I
5
IO
:
: b
I5
6Gu-
Figure 4. Binding of c3H] PM to reticulocyte ribosomes. (3H) PM (lo-7 M, 6 x lo4 cpm/reaction) was added at O" to 3 units (A260m,,) of ribosomes in 1 ml of the samebuffer as in the sucrose gradient. This solution was layered on the sucrose gradient and analyzed as described in Materials and Methods. A) control ribosomes, B) prior to addition of PM, control ribosomes were treated with 0.15 ug crystallin pancreatic RNasefor 10 min at O", C) ribosomes from NaF-treated cells (prepared as in Figure 2). 6
VoL41,No.
1,197O
BIOCHEMICAL
suggesting that only a fraction
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of the 80s ribosomes bind PM. At 37O
binding to the polyribosomes can also be found.
Increasing the amount of
80s ribosomes by treatment of the polyribosomes with RNasedoes not increase the amount of PMbound at O". which produces an equivalent initiation at O".
On the other hand, NaF treatment of reticulocytes amount of 80s ribosome in the lysate by blocking
of protein synthesis, 14y15 greatly As found by Colombo, -L' et al
increases the amount of PMbound
l6 NaF treatment decreased ribosome ,subunits;
PM binding to the 40s subunit is similarly
markedly decreased.
The single ribosomes formed by treatment of reticulocytes produced by the run-off
with NaF are
of ribosomes from polyribosomes during the course of
peptide chain completion and release, and presumably lack mRNA,peptide and possibly protein factors.
On the other hand, RNase-produced single ribosomes
carry a fragment of mRNA,peptide and possibly protein
factors.
It
is likely
that the marked increase in binding of 3H-PM found with the NaF-produced ribosomes is due to the availability
of a binding site for PMon such ribosomes
(present on the 40s subunit) which is lacking, hindrance or allosteric
change, on the RNase-producedribosomes.
possible that once the initiation has taken place, the particular of initiation
possibly because of steric It is
complex is formed and peptide-bond formation PMbinding site responsible for the inhibition
is no longer accessible to the antibiotic.
References 1. 2. 3. 4. 5. 6. 7. 8. 9.
Colombo, B., Felicetti, L., and Baglioni, C. (1966), Biochim. et Biophys. Acta, 119:109. Felicetti, L., Colombo, B., and Baglioni, C. (1966), Biochim. et Biophys. Acta, 119:lZO. Bhuyan, B.K. (1967), Biochem. Pharmacol., 16:1411. Cundliffe, E., and McQuillan, K. (1967), JTMol. Biol., 30~137. Cohen, L.B., Herner,‘A.E., and Goldberg, I.H. (1969), Biochemistry, 8:1312. Cohen, L.B., Goldberg, I.H., and Herner, A.E. (1969), Biochemistry, 8:1327. Cohen, L.B., and Goldberg, I.H. (1967), Biochem. Biophys. Res. Cornmu:., 29:617. Bollurn, F.J. (1966) in Procedures in Nucleic Acid Research, Harper and Rowe, New York, p. 296. Hardesty, B., Miller, R., and Schweet, R. (1963), Proc. Natl. Acad. Sci., -50:924. 7
VoL41,No.
10. 11. 12. 13. 14. 15. 16.
1,197O
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Hardesty, B., Hutton, J.J., and Arlinghaus, R. (19633, Proc. Natl. Acad. Sci., 50:1078. JayarEn, J., and Goldberg, I.H. (1968), Biochemistry, 7:418. Marks, P.A., Burka, E.R., Conconi, F.M., Perl, W., and Ryfkind, R.A. (1965), Proc. Natl. Acad. Sci., 53:1437. Felicetti, L., and Lipmann, F. (1968), Arch. Biochem. Biophys., 125:548. Lin, S., Mosteller, R.D., and Hardesty, B. (1966), J. Mol. Biol.>l:Sl. Ravel, J.M., Mosteller, R.D., and Hardesty, B. (1966), Proc. Natl.Acad. Sci., 56:701. Colombc B., Vesco, C., and Baglioni, C. (1968), Proc. Natl. Acad. Sci., -61:651.
8
BIOCHEMICAL
1, 1970
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
AN EFFECT OF PACTAMYCIN ON THE INITIATION
OF
PROTEIN SYNTHESIS IN RETICULOCYTES* John S. Macdonald Department.of Beth Israel Received
Medicine, Hospital,
and Irving
H. Goldberg
Harvard Medical School and the Boston, Massachusetts 02215
July 31, 1970
Summary: Low levels of pactamycin (about 10m6 M) stop polypeptide synthesis by intactreticulocytes and their lysates after a lag of about two minutes. This effect which is similar to that of poly A, an inhibitor of polypeptide chain is not additive to that of poly A. Cells which have been preinitiation, treated with NaF and must form new polypeptide chains are more sensitive to pactamycin than cells which are able to complete pre-existing chains. High levels of pactamycin (&1O-5 M) inhibit elongation in addition to initiation. ‘H-pactamycin binds to 80s ribosomes and the 40s subunits at 0’. Monosomes very effectively, produced by NaF treatment of reticulocytes bind 5H-pactamycin whereas those produced by RNase treatment of polyribosomes do not. Pactamycin whole
cells
shown
that
(PM) is
and extracts PM binds
structure
a potent
of the
phenylalanyl-tRNA,
antibiotic
of procaryotic
to the
initiator
and eucaryotic
complex
N-formylmethionyl-tRNA,
(with
of polypeptide
synthesis
of inhibition
does not effect
interfer
is with
also
data,
the conversion
coupled of rabbit
of soluble,
greater
newly
the
N-aaetylresulting
synthesis
in
itself,
its
PM inhibition is greater has started;
at low Mg++ concentrations.’
bond formation
the
PM, however,
as judged
by its
lack
the finding
reticulocyte synthesized
of Colombo,
polyribosomes protein,
al -et 2)
’ that
to 80s ribosomes
suggest
that
this
PM induces with
antibiotic
* This work U.S. Public
of
2,4,5,7
reaction. with
and affects
Furthermore,
polypeptide
in
It has been
by N-acetylphenylalanyl-tRNA before
peptide
on the puromycin These
release
initiated
is present
of --E. coli
as “initiator”-tRNA),
at low Mg++ concentrations.5-7
synthesis
cells.l-5
subunit
disassociation
extent
of protein
30s ribosomal
or polylysyl-tRNA
when the antibiotic
inhibitor
was supported by grants from the National Institutes Health Service (GM 12573) and the American Cancer
of Health, Society.
the may
Vol. 41,No.
affect
BIOCHEMICAL
1, 1970
the initiation
of polypeptide
experiments (M. Stewart,
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
synthesis.
I. Yanowitz and I.H.
This view finds support in Goldberg, in preparation),
where low levels of PM (about 10m6M, dependent on the number of ribosomes) rapidly
and almost quantitatively
in lysates from rabbit
produce single ribosomes from polyribosomes
reticulocytes
with the release of completed hemoglobin
chains. Wenow show that low concentrations initiation inhibit
in reticulocytes elongation.
and their
of PMmainly affect
lysates,
polypeptide
chain
but that higher concentrations
also
Furthermore, the ribosomal binding site for PM (at O" and
10-7 M PM) is absent in ribosomes bearing a fragment of mRNA,such as are produced by RNasetreatment of polyribosomes. Materials
and Methods:
and cell-free
The preparation
and incubation of rabbit
lysates were as described by Colombo, et al.,'
lysate system the incorporation acid-precipitable
of ( l4C) valine
reticulocytes
except that in the
into hot trichloroacetic
protein was assayed by the filter
paper disc method described
by Bollum8.
The binding of (3H) PM, prepared as previously
reticulocyte
ribosomes was measured on 15-30% sucrose density gradients contain-
ing 0.01 M Tris,
pH 7.4, 0.01
in the Spinco rotor collected
SW41
KCl, and 0.0015 M MgClz.
analysis as previously
Results and Discussion:
synthesis by reticulocyte
which the particular the incorporation
lysate
with the initiation
varies with the degree to
depends on the formation of new chains for
of labelled amino acids.
Figure 1 shows that in the presence
proceeds at control
rates for almost two
minutes, at which time there is an abrupt cessation of further also shows that 10s6 PM gives essentially
inhibition
as does poly A.
were
absorbance recorder
lysates by interfering
of poly A the synthesis of polypeptide
figure
centrifugation
have shown that poly A inhibits
The extent of inhibition system
to
described.6
Hardesty, et al., '*lo
of new hemoglobin chains.
After
41,000 RPMfor 90 min. at 4O, fractions
from the bottom of the tube through a Gilford
for radioactivity
polypeptide
at
M
reported,6
the same level and kinetics
Furthermore, when poly A and 10-6
2
synthesis.
PM
This of
are both present,
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 41, No. 1, 1970
SPARS
0
2
4
6
8
+ POLY A
10
protein synthesis and poly A on cell-free Figure 1. Effect of antibiotics The incorporation of (14C) valine (35 uc/umole ) into protein by reticulocy t e The volume of the reaction lysates was followed as previously described.l . mixture was 500 ul; 75 1.11was removed for assay at each _. of . ^the indicated times. Poly A (100 ug/ml), PM and sparsomycin were added tram tne start ot the incubation, as indicated
the pattern additive
and extent of inhibition
effect
is that of either one alone; there is no
suggesting that both agents are exerting
the sameoverall
step in protein
maximal actions at
synthesis.
During the two minute lag in onset of the inhibitory
action of PM (and
poly A) nascent peptide chains are presumably being completed and released fron the ribosome. continuously,
In reticulocytes
or lysates where new chains are being formed
the lag in onset of the PM (10-6 M) inhibition
although the final
extent of inhibition
increases as the number of rounds of
synthesis of chains increases in the control. the inhibitory
effect
is always found,
Since PMbinds to the ribosome,
depends on both the PMand ribosome concentrations. 3
Vol.41,No.
BIOCHEMICAL
1, 1970
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Increasing the number of ribosomes but not the amount of soluble protein decreases the PM effect A very different
(Cohen and Goldberg, unpublished results). result
is found with sparsomycin, which is an inhibitor
of peptide bond formation.ll
At the low level of sparsomycin (6 x 10-8 M)
used in Figure 1, the rate of protein start
and this effect
is additive
synthesis is slowed down from the very
with that due to poly A.
(Figure 2A) that at higher concentrations protein biotic
synthesis is also inhibited, polypeptide
rate of
suggesting that at high levels of anti-
to this inhibition.
found at high PM concentrations location
of PM (10-S M), the initial
chain elongation is also affected.
does not contribute
It should be noted
The inhibition
probably results
since peptide bond formation
As expected, poly A of chain elongation
from an effect
(the puromycin reaction)
on transremains intact
(I. Yanowitz and I.H. Goldberg, unpublished data).
A. 3000
CONTROL
8.
Na F
-
2500
5ooc 0
2
4
6
8
10
of NaF-treated and untreated reticulocytes to PM. Figure 2. Sensitivity Reticulocytes were incubated as previously described1 without (A) or with (B) 0.01 M NaF for 30 min. at 37O. After addition of equal volumes of cold saline, the cells were washed four times with the samebuffer prior to reincubation under standard conditions (0.8 nc of 14C-amino acid mix (1.5 mc/mg) per reaction)l in the presence or absence of PM. At the indicated times 100 1.11aliquots were removed for the analysis of 14C incorporation into pretein. 4
Vol. 41, No. 1, 1970
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Figure 2 also shows that the pretreatment
of reticulocytes
with NaF, which
converts most of the polyribosomes to single ribosomes,12 increases the sensitivity
of polypeptide
synthesis is almost entirely chains.
synthesis to PM. In this system, polypeptide dependent on the initiation
Similar results have been found with protein
prepared from NaF treated cells
(M. Stewart,
of new hemoglobin synthesis in lysates
I. Yanowitz and I.H.
Goldberg,
unpublished results). In a system (polyphenylalanine physiological affects
synthesis) that is not dependent on the
mechanismfor the initiation
the overall
of protein
synthesis,
PM (10e5 M)
synthesis only to a small degree and, as with sparsomycin,
acts mainly by decreasing the initial
M/W
rate of synthesis (Figure 3).
TES
Figure 3. Effect of PM on poly U-dependent polyphenylalanine synthesis. S-100 and ribosomal fractions were obtained by centrifugation of a 1:l reticulocyte lysate at 105,OOOxgfor 2 hours. NH4Cl washed ribosomes were prepared as described by Felicetti and Lipmann.13 Polyphenylalanine synthesis was followed by incubating the following in a volume of 200 1.11: 40 ug E. coli (14C) phe-tRNA (330 cpm/ug), 10 umoles Tris, pH 7.4, 16 umoles KCl, 0.8or0.8 umoles GTP, 2 units (A260mu) 1.6 umoles MgC12, 3.2 umoles dithiothreitol, ribosomes, 40 ug poly U, and 30 ul S-100. At the indicated times 30 ul aliquots were removed for assay on filter paper discs.8 PM (lo-5 M) was added as indicated. In the absence of poly U, 200 cpm were incorporated at 15 min. 5
Vol. 41,No.
BIOCHEMICAL
1, 1970
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
PM binds at O" to the 80s ribosome and the 40s ribosomal subunit of the reticulocyte,
but not the polyribosome (Figure 4).
The specific
activity
of
PMbound to 40s subunits is much higher than that bound to 80s ribosomes,
200
-
ii’iI 020 ?5-J ’\:I :? -I 0.10
\
d
I
5
IO
15
I
5
IO
:
: b
I5
6Gu-
Figure 4. Binding of c3H] PM to reticulocyte ribosomes. (3H) PM (lo-7 M, 6 x lo4 cpm/reaction) was added at O" to 3 units (A260m,,) of ribosomes in 1 ml of the samebuffer as in the sucrose gradient. This solution was layered on the sucrose gradient and analyzed as described in Materials and Methods. A) control ribosomes, B) prior to addition of PM, control ribosomes were treated with 0.15 ug crystallin pancreatic RNasefor 10 min at O", C) ribosomes from NaF-treated cells (prepared as in Figure 2). 6
VoL41,No.
1,197O
BIOCHEMICAL
suggesting that only a fraction
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of the 80s ribosomes bind PM. At 37O
binding to the polyribosomes can also be found.
Increasing the amount of
80s ribosomes by treatment of the polyribosomes with RNasedoes not increase the amount of PMbound at O". which produces an equivalent initiation at O".
On the other hand, NaF treatment of reticulocytes amount of 80s ribosome in the lysate by blocking
of protein synthesis, 14y15 greatly As found by Colombo, -L' et al
increases the amount of PMbound
l6 NaF treatment decreased ribosome ,subunits;
PM binding to the 40s subunit is similarly
markedly decreased.
The single ribosomes formed by treatment of reticulocytes produced by the run-off
with NaF are
of ribosomes from polyribosomes during the course of
peptide chain completion and release, and presumably lack mRNA,peptide and possibly protein factors.
On the other hand, RNase-produced single ribosomes
carry a fragment of mRNA,peptide and possibly protein
factors.
It
is likely
that the marked increase in binding of 3H-PM found with the NaF-produced ribosomes is due to the availability
of a binding site for PMon such ribosomes
(present on the 40s subunit) which is lacking, hindrance or allosteric
change, on the RNase-producedribosomes.
possible that once the initiation has taken place, the particular of initiation
possibly because of steric It is
complex is formed and peptide-bond formation PMbinding site responsible for the inhibition
is no longer accessible to the antibiotic.
References 1. 2. 3. 4. 5. 6. 7. 8. 9.
Colombo, B., Felicetti, L., and Baglioni, C. (1966), Biochim. et Biophys. Acta, 119:109. Felicetti, L., Colombo, B., and Baglioni, C. (1966), Biochim. et Biophys. Acta, 119:lZO. Bhuyan, B.K. (1967), Biochem. Pharmacol., 16:1411. Cundliffe, E., and McQuillan, K. (1967), JTMol. Biol., 30~137. Cohen, L.B., Herner,‘A.E., and Goldberg, I.H. (1969), Biochemistry, 8:1312. Cohen, L.B., Goldberg, I.H., and Herner, A.E. (1969), Biochemistry, 8:1327. Cohen, L.B., and Goldberg, I.H. (1967), Biochem. Biophys. Res. Cornmu:., 29:617. Bollurn, F.J. (1966) in Procedures in Nucleic Acid Research, Harper and Rowe, New York, p. 296. Hardesty, B., Miller, R., and Schweet, R. (1963), Proc. Natl. Acad. Sci., -50:924. 7
VoL41,No.
10. 11. 12. 13. 14. 15. 16.
1,197O
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Hardesty, B., Hutton, J.J., and Arlinghaus, R. (19633, Proc. Natl. Acad. Sci., 50:1078. JayarEn, J., and Goldberg, I.H. (1968), Biochemistry, 7:418. Marks, P.A., Burka, E.R., Conconi, F.M., Perl, W., and Ryfkind, R.A. (1965), Proc. Natl. Acad. Sci., 53:1437. Felicetti, L., and Lipmann, F. (1968), Arch. Biochem. Biophys., 125:548. Lin, S., Mosteller, R.D., and Hardesty, B. (1966), J. Mol. Biol.>l:Sl. Ravel, J.M., Mosteller, R.D., and Hardesty, B. (1966), Proc. Natl.Acad. Sci., 56:701. Colombc B., Vesco, C., and Baglioni, C. (1968), Proc. Natl. Acad. Sci., -61:651.
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