Nucleic Acids Research
Volume 5 Number 3 March 1978
66 Studies on ribosome structure and interactions near the m2A m2A sequence
Pallaiah Thammana and Charles R. Cantor
Departments of Chemistry and Biological Sciences, Columbia University, New York, NY 10027, USA Received 29 December 1977 ABSTRACT
Antibodies raised againsa N6,N6-dimethyl adenosine were used to study the environment and role of the m2A m2A sequence in the E. coli ribosome. It is observed that this sequence is exposed on the surface of isolated 30S subunits, but becomes inaccessible for IgG interaction upon heat activation. The m6A sequence is also inaccessible for IgG interaction in 70S ribosomes or 30S subunits immediately after dissociation of 70S particles. The presence of IgGs results in a significant inhibition of IF3 binding to unactivated 30S particles. IF3 binding to activated 30S subunits is unaffected by the IgGs. Crosslinking of 30S proteins S18 and S21 with the bifunctional phenylene dimaleimide reagents results in a reduction in the extent of 30S-IgG interaction. From what is already known about the location of S18, S21 and the IF3 sequence is located close to binding site, it is suggested that the m6A the initiator tRNA binding site of the 30S subunit during initiation of protein synthesis.
mgA
mgA
INTRODUCTION
Much recent evidence suggests that a region in the 30S ribosomal subunit near the 3' end of 16S RNA plays an important role in the function of E. coli
ribosomes.
Presence of a methylated sequence m6A m6A near the 3' end of
The 16S RNA is responsible for sensitivity to the antibiotic kasugamycin. inte15cur inntesqec 6 in 6 6A CCUG CGn near the 3' end. the sequence m2A only m62A the 16S RNA occurs m2A
Kasugamycin resistant mutants lack the enzyme which methylates this AACCUG sequence, and ribosomes from these mutants are resistant to the antibiotic in vitro. ' Kasugamycin is an inhibitor of initiation of protein synthesis, This and specifically inhibits binding of initiator tRNA to ribosomes. ' suggests that the m6A m6A sequence is located close to a functional site of the 30S subunit during initiation complex formation. We have employed antibodies raised against protein conjugated m6A for studies on the structural organization of the 30S subunit near the m6A m6A sequence. The antibodies used were originally isolated and characterized antibodies by Glitz and his colleagues. Politz and Glitz showed that interact with 30S subunits isolated from a kasugamycin sensitive strain, and
m9A
C Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England
805
Nucleic Acids Research that this interaction can be visualized by electron microscopy.15 In this article we show how these antibodies can probe different conformational states of 16S RNA in the 30S subunits. We have observed that the m2A m2A sequence exists in two distinct environments in different conformational states of the 30S subunits. The functional implications of these structural differences will be discussed. MATERIALS AND METHODS
Bacterial strains. E. coli strain PR7 and its kasugamycin resistant derivative TPR 201 were obtained from Prof. Julian Davies. Log phase E. coli MRE 600 (RNaseI-) cells were purchased from Microbiological Research Establishment, Porton, England. Chemicals. N-ethyl maleimide (NEM), N-phenyl maleimide (PM), N-phenylene o-dimaleimide (o-PDM), and N-phenylene p-dimaleimide (p-PDM) were purchased frcm Aldrich Chemical Company. Kasugamycin hydrochloride was a gift from Bristol Laboratories. Streptomycin sulfate was from Sigma Chemical Company. 14C-Formaldehyde (sp. act. 44mc/mmole) was purchased from New England Nuclear. Sodium Borohydride was from Alfa Products. 14C-Phenylalanine and 3H-methionine were purchased from New England Nuclear. Poly U was purchased from Sigma, and unfractionated tRNA was from Boeringer Mannheim Biochemicals. The following pH 7.6 Tris-HCl buffers were used: Name
Tris-HCl
Mg 2+ acetate
TMAI
1OmM
1OmM
30mM
A
1OmM
B
IgG
lOmM 1OmM 20mM
1OmM 1OmM 0. 3mM!
C
40mM
40mM
D
20mM
5mM
E
20mM
5mM
1M 1.5M 30mM 150mM 400mM 150mM 1OOmM
F
20mM
1mM
G
2OmM
H
1OmM 5OmM
15mM 1.5mM
TMAII
J
0
5mM
NH 4C
8-mercaptoethanol 6mM 6mM 6mM
6mM 0
24mM 6mM
6mM
0OOmM 0OOmM
6mM
30mM
6mM
50mM
6mM
6mM
Preparation of ribosomes and subunits. Ribosomes were isolated from frozen cell masses of E. coli MRE 600 by alumina grinding in TMAI buffer. Ribosomes were routinely high salt washed three times as described below. The crude ribosome pellet obtained after breaking open the cells was dissolved in 806
Nucleic Acids Research TMAI and layered
10% sucrose solution in buffer A, and centrifuged for
on a
17.5 hours at 25,000
rpm
in
dissolved in buffer B and centrifuged for Ribosomal pellets
Ti rotor. 10%
sucrose
in buffer A
as
The ribosomal pellets
42.1 rotor.
a
45,000
two hours at
rpm
were
layered
The resulting ribosomes
described above.
and centrifuged in
an
10-25% discontinuous
over a
in
a
50.2
dissolved in TMAI and sedimented through
were
dissolved in TMAI and frozen at -700. Ribosomes were dissociated by dialysis against TMAII buffer. ribosomes
then
were
SW27 rotor at 23,000
rpm
sucrose
were
Dissociated
gradient in TMAII,
for 17 hours. 16
50S subunits
moved almost to the bottom of the centrifuge tube under these conditions, and a
layer of 30S subunits could be
Both 30S and 50S subunit layers tion
brought
was
were
above 50S subunits by Tyndall scattering.
pooled carefully, and the Mg
Pooled subunits
lOmM.
up to
seen
were
concentra-
pelleted, dissolved in
TMAI, and frozen in small aliquots. Ribosomes and subunits were isolated from kasugamycin resistant strain TPR 201 essentially as described above. We did not observe a significant difference in the IgG interaction with low salt
washed 30S subunits prepared by conventional methods paper,
only the high salt washed 30S
Purified ribosome couples by
a
were
salt washed
once
The
used.
isolated from slow cooled MRE 600 cells
modification of the procedure of Noll et
Ms. Elizabeth Matthews. were
were
compared to the sub-
In all the experiments presented
units prepared by methods mentioned above.
in this
as
al.,1
and kindly provided by
procedure used differed from Noll's in that, 70S
with 1.5M NH4C1, and
by zonal ultracentrifugation in
a
5mM Mg
pure
ribosome couples
were
prepared
acetate containing buffer.
Concentration of ribosomes and ribosomal subunits were determined from absorbance measurements at 260 nm. (.A260 30S = 66 pmoles; lA 260 70S = 22 pmoles). Anti
m6A 2
antibodies
Anti m2A IgG preparations used in this study were the generous gift of Dr. Dohn Glitz.
Two different preparations of purified IgGs designated as
Batch III and Batch D were used in experiments presented here. Details of immunization and purification of these IgGs are published by Politz and Glitz.
Politz and Glitz estimated that the Batch D IgGs we used had a
binding capacity of about 8 times either Batch III
or Batch D
IgGs
and stored at -70° until use. ribosomes
more than the Batch III IgGs. were
m6A
Routinely
dialysed overnight against IgG buffer,
Required amounts of IgGs were added, and
were incubated at 370 as indicated in
figure legends.
The amount
807
Nucleic Acids Research of IgGs required for optimal dimer formation
each Batch of IgGs. Lowry et al.
Protein concentration
were
of
IgGs
reductively methylated according to the general procedures
described by Pon et al. dialysed
21
measured by the method of
1 A280 of IgGs corresponds to 840 pg of protein.
Reductive methylation IgGs
determined separately for
was
was
About 3A
units of Batch D IgGs
period of 2 hours with 3 changes of buffer. 14C-Formaldehyde (aqueous solution)
was
Reactions
sodium borohydride
were
a
final
Two 204l aliquots of
concen-
a
0.5%
then added with 1 minute intervals, and the reduction
carried out for 15 minutes at 00.
Samples
were
then dialysed against IgG
buffer overnight until all unreacted formaldehyde activity of labelled IgGs
was
removed.
was
Specific
449 dpm/ig protein.
Incubation of ribosomes and subunits with IgGs and general procedures of Zamir et al.
The
a
carried out at 00.
were
added to the IgGs to
tration of 3mM and incubated for 30 seconds.
tion.
fast
were
280
against 50mM sodium borate buffer pH 9 containing 200mM KC1 for
30S subunits (200-300 A 260units/mi)
were were
sucrose
followed for heat activa-
mixed with
of buffer C, and incubated at 400 for 30 minutes.
gradient analysis
an
equal volume
Activated subunits
were
chilled on ice, and immediately used for IgG interaction.
Activated
or
unactivated 30S subunits
were
incubated with the IgGs
indicated in the figure legends in buffer D at 37° for 15 minutes. end of incubation, the 1004l samples were chilled
layered in
an
over
10-30%
sucrose
gradients in buffer E.
SW 50.1 rotor at 32,000
rpm
Radioactivity
was
were
At the
ice, and immediately Gradients
for 4.5 hours at 4
dripped manually, 3-4 drop fractions at 260 nm.
on
as
.
were
Gradients
centrifuged were
collected, and absorbance measured
measured by liquid scintillation counting in
Triton X-100 cocktail. For the dissociation-reassociation experiments, 70S ribosome couples were
incubated at 370
chilled
on
1M Mg
acetate.
Gradients
analysed
as
indicated, in buffer F.
ice and immediately brought were
as
Samples
were
centrifuged in
analysed an
up
After incubation, samples
were
to 15mM Mg
on sucrose
concentration by adding gradients in buffer G.
SW 50.1 rotor at 32,000 rpm for 3 hours, and
described above.
Cross-linking of 30S subunits 30S subunits were fast dialysed against a buffer containing 10mM Trisacetate for a period of 1.5 hours with 5 HC1 pH 7.8, 50mM KC1, and lmM Mg
changes of 300 ml each.
808
Dialysed 30S subunits freed of mercaptoethanol were
Nucleic Acids Research incubated with a cross-linking control reagent for 30 minutes at 300. PM and o-PDM were freshly dissolved in acetone while p-PDM was dissolved in dimethyl formamide (due to low solubility in acetone) at a concentration of lOmM. The reagents were added to the 30S subunits at a final concentration of 0.4 mM exactly as described by Chang and Flaks.23 Under our conditions, the ratio of subunits to reagent was 1:30. After a 30 minute reaction, a-mercaptoethanol was added to a final concentration of lOmM, and incubations continued for 10 minutes. Samples were dialysed against buffer H. Gel electrophoresis
Cross-linked proteins were analysed by polyacrylamide gel electrophoresis at pH 4.5 as described by Hardy et al. 4 30S proteins were extracted by the RNase method of Nashimoto et al. 25 Two different gel systems were used for SDS polyacrylamide gel electrophoresis. 10% gels were used in a Weber's system, 6 and 13.5% gels were employed in a Laemmli system. 27 Samples for electrophoresis were prepared by heating 30S subunits at 650 for 10 minutes in 1% SDS, 2% B-mercaptoethanol, 4mM EDTA and 4M Urea. Gels were scanned by
ISCO gel scanner model 659, and absorbance recorded by ISCO Model UA-5 absorbance monitor.
Binding
of
C-labelled IF3 to 30S 14C-labelled purified initiation factor IF3 was a generous gift from Drs. Barry Cooperman and Marianne Grunberg-Manago. 30S subunits were incubated with the labelled IF3 in buffer E at 370 for 15 minutes. ° Incubated samples were layered on sucrose gradients in the incubation buffer and analysed as described for 30S-IgG dimers except that the samples were processed directly for scintillation counting unless otherwise stated. tRNA binding assays
R17 RNA dependent fMet-tRNA binding to 30S subunits was done according R17 RNA was isolated from R17 phage grown
to general published procedures.
according to a procedure described by Dr. R. F. Gestland (personal communication). 0.73 A260 units of MRE 600 30S subunits (48 pmoles) were incubated with 0.44 A 260 units of R17 RNA, 0.38 A 3H fMet-tRNA (19.8 260 units of pmoles; specific activity 11,005 dpm/pmole), optimized amounts of ammonium sulfate fractionated crude initiation factors, 28 and 1.6 mM GTP. tRNA binding reactions were carried out in a 5Ol volume of buffer J, at 370 for 15 minutes. At the end of incubation samples were chilled on ice, and diluted with 1.5 ml of cold buffer J. Samples were filtered on Millipore Type HA filters followed by 3 washes with the same buffer.
Dried filters
were
counted in 809
Nucleic Acids Research PPO-POPOP scintillation fluid.
The counting efficiency was 21%.
Poly U dependent Phe-tRNA binding to 30S subunits was carried out
according to a procedure similar to that described above. 0.74 A260 units of 30S subunits (49 pmoles) were incubated with 0.84 A260 units of Poly U, and 1 A 260 unit of C Phe-tRNA (38 pmoles; specific activity 461 dpm/pmole) 30 at 0° for 60 minutes. Reactions were in 100 4l of 10 mM Tris-HCl buffer
(pH 7.2) containing 60 mM NH 4 Cl, 10 mM Mg acetate, and 6 mM B-mercaptoethanol. tRNA bound to 30S subunits was determined as described above. The counting efficiency was 74%. tRNAs were charged using procedures kindly provided by Dr. A. E. Johnson. RESULTS Interaction of antibody with 30S particles Sucrose gradient centrifugation was used to study the interaction of
m6A
antibodies with 30S subunits isolated from E. coli strain MRE 600. Fig. la shows a typical yield of 30S dimer formation upon incubation of 30S subunits with the IgGs. The control experiment shown in Fig. lb, demonstrates that the methyl deficient 30S particles from kasugamycin resistant strain TPR 201, fail to form dimers; only free 30S particles and IgGs are found after incubation. No dimers are seen with 30S subunits that have been incubated under similar conditions in the absence of IgGs. We have also failed to observe dimers upon incubation of 30S with non-immune IgGs (data not shown). These experiments demonstrate the specificity of the antibody reaction resulting in dimer formation. m6A Although the antibodies recognize the m6A 2 2 sequence in the isolated 30S subunits as shown in Fig. la, they do so only with the inactive conformation of 30S. Zamirand coworkers observed that heat activation of 30S subunits characteristically enhances the binding of 50S subunits and binding of transfer RNA in the presence of messenger RNA. ' In our hands, heat activation produced a 2-3 fold stimulation in R17 RNA dependent fMet-tRNA binding to 30S subunits and a 24 fold stimulation in Poly U dependent Phe-tRNA binding to 30S subunits. See Table I. Incubation of IgGs with 30S subunits yields no heat activated according to the procedures of Zamir et al., is rendered inaccessidimers (Fig. lc). Apparently, the methylated sequence ble for dimer formation upon heat activation of 30S subunits. To demonstrate that direct interaction of IgGs with the subunits is required for dimer formation, we employed radioactive IgGs prepared by reductive methylation with 14C-formaldehyde as described in materials and methods. 810
Data
presented in Fig. 2 demonstrate that dimer formation involves
Nucleic Acids Research (a) .3
.2
.1
(b) .3
E .2 0
Volume 5 Number 3 March 1978
66 Studies on ribosome structure and interactions near the m2A m2A sequence
Pallaiah Thammana and Charles R. Cantor
Departments of Chemistry and Biological Sciences, Columbia University, New York, NY 10027, USA Received 29 December 1977 ABSTRACT
Antibodies raised againsa N6,N6-dimethyl adenosine were used to study the environment and role of the m2A m2A sequence in the E. coli ribosome. It is observed that this sequence is exposed on the surface of isolated 30S subunits, but becomes inaccessible for IgG interaction upon heat activation. The m6A sequence is also inaccessible for IgG interaction in 70S ribosomes or 30S subunits immediately after dissociation of 70S particles. The presence of IgGs results in a significant inhibition of IF3 binding to unactivated 30S particles. IF3 binding to activated 30S subunits is unaffected by the IgGs. Crosslinking of 30S proteins S18 and S21 with the bifunctional phenylene dimaleimide reagents results in a reduction in the extent of 30S-IgG interaction. From what is already known about the location of S18, S21 and the IF3 sequence is located close to binding site, it is suggested that the m6A the initiator tRNA binding site of the 30S subunit during initiation of protein synthesis.
mgA
mgA
INTRODUCTION
Much recent evidence suggests that a region in the 30S ribosomal subunit near the 3' end of 16S RNA plays an important role in the function of E. coli
ribosomes.
Presence of a methylated sequence m6A m6A near the 3' end of
The 16S RNA is responsible for sensitivity to the antibiotic kasugamycin. inte15cur inntesqec 6 in 6 6A CCUG CGn near the 3' end. the sequence m2A only m62A the 16S RNA occurs m2A
Kasugamycin resistant mutants lack the enzyme which methylates this AACCUG sequence, and ribosomes from these mutants are resistant to the antibiotic in vitro. ' Kasugamycin is an inhibitor of initiation of protein synthesis, This and specifically inhibits binding of initiator tRNA to ribosomes. ' suggests that the m6A m6A sequence is located close to a functional site of the 30S subunit during initiation complex formation. We have employed antibodies raised against protein conjugated m6A for studies on the structural organization of the 30S subunit near the m6A m6A sequence. The antibodies used were originally isolated and characterized antibodies by Glitz and his colleagues. Politz and Glitz showed that interact with 30S subunits isolated from a kasugamycin sensitive strain, and
m9A
C Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England
805
Nucleic Acids Research that this interaction can be visualized by electron microscopy.15 In this article we show how these antibodies can probe different conformational states of 16S RNA in the 30S subunits. We have observed that the m2A m2A sequence exists in two distinct environments in different conformational states of the 30S subunits. The functional implications of these structural differences will be discussed. MATERIALS AND METHODS
Bacterial strains. E. coli strain PR7 and its kasugamycin resistant derivative TPR 201 were obtained from Prof. Julian Davies. Log phase E. coli MRE 600 (RNaseI-) cells were purchased from Microbiological Research Establishment, Porton, England. Chemicals. N-ethyl maleimide (NEM), N-phenyl maleimide (PM), N-phenylene o-dimaleimide (o-PDM), and N-phenylene p-dimaleimide (p-PDM) were purchased frcm Aldrich Chemical Company. Kasugamycin hydrochloride was a gift from Bristol Laboratories. Streptomycin sulfate was from Sigma Chemical Company. 14C-Formaldehyde (sp. act. 44mc/mmole) was purchased from New England Nuclear. Sodium Borohydride was from Alfa Products. 14C-Phenylalanine and 3H-methionine were purchased from New England Nuclear. Poly U was purchased from Sigma, and unfractionated tRNA was from Boeringer Mannheim Biochemicals. The following pH 7.6 Tris-HCl buffers were used: Name
Tris-HCl
Mg 2+ acetate
TMAI
1OmM
1OmM
30mM
A
1OmM
B
IgG
lOmM 1OmM 20mM
1OmM 1OmM 0. 3mM!
C
40mM
40mM
D
20mM
5mM
E
20mM
5mM
1M 1.5M 30mM 150mM 400mM 150mM 1OOmM
F
20mM
1mM
G
2OmM
H
1OmM 5OmM
15mM 1.5mM
TMAII
J
0
5mM
NH 4C
8-mercaptoethanol 6mM 6mM 6mM
6mM 0
24mM 6mM
6mM
0OOmM 0OOmM
6mM
30mM
6mM
50mM
6mM
6mM
Preparation of ribosomes and subunits. Ribosomes were isolated from frozen cell masses of E. coli MRE 600 by alumina grinding in TMAI buffer. Ribosomes were routinely high salt washed three times as described below. The crude ribosome pellet obtained after breaking open the cells was dissolved in 806
Nucleic Acids Research TMAI and layered
10% sucrose solution in buffer A, and centrifuged for
on a
17.5 hours at 25,000
rpm
in
dissolved in buffer B and centrifuged for Ribosomal pellets
Ti rotor. 10%
sucrose
in buffer A
as
The ribosomal pellets
42.1 rotor.
a
45,000
two hours at
rpm
were
layered
The resulting ribosomes
described above.
and centrifuged in
an
10-25% discontinuous
over a
in
a
50.2
dissolved in TMAI and sedimented through
were
dissolved in TMAI and frozen at -700. Ribosomes were dissociated by dialysis against TMAII buffer. ribosomes
then
were
SW27 rotor at 23,000
rpm
sucrose
were
Dissociated
gradient in TMAII,
for 17 hours. 16
50S subunits
moved almost to the bottom of the centrifuge tube under these conditions, and a
layer of 30S subunits could be
Both 30S and 50S subunit layers tion
brought
was
were
above 50S subunits by Tyndall scattering.
pooled carefully, and the Mg
Pooled subunits
lOmM.
up to
seen
were
concentra-
pelleted, dissolved in
TMAI, and frozen in small aliquots. Ribosomes and subunits were isolated from kasugamycin resistant strain TPR 201 essentially as described above. We did not observe a significant difference in the IgG interaction with low salt
washed 30S subunits prepared by conventional methods paper,
only the high salt washed 30S
Purified ribosome couples by
a
were
salt washed
once
The
used.
isolated from slow cooled MRE 600 cells
modification of the procedure of Noll et
Ms. Elizabeth Matthews. were
were
compared to the sub-
In all the experiments presented
units prepared by methods mentioned above.
in this
as
al.,1
and kindly provided by
procedure used differed from Noll's in that, 70S
with 1.5M NH4C1, and
by zonal ultracentrifugation in
a
5mM Mg
pure
ribosome couples
were
prepared
acetate containing buffer.
Concentration of ribosomes and ribosomal subunits were determined from absorbance measurements at 260 nm. (.A260 30S = 66 pmoles; lA 260 70S = 22 pmoles). Anti
m6A 2
antibodies
Anti m2A IgG preparations used in this study were the generous gift of Dr. Dohn Glitz.
Two different preparations of purified IgGs designated as
Batch III and Batch D were used in experiments presented here. Details of immunization and purification of these IgGs are published by Politz and Glitz.
Politz and Glitz estimated that the Batch D IgGs we used had a
binding capacity of about 8 times either Batch III
or Batch D
IgGs
and stored at -70° until use. ribosomes
more than the Batch III IgGs. were
m6A
Routinely
dialysed overnight against IgG buffer,
Required amounts of IgGs were added, and
were incubated at 370 as indicated in
figure legends.
The amount
807
Nucleic Acids Research of IgGs required for optimal dimer formation
each Batch of IgGs. Lowry et al.
Protein concentration
were
of
IgGs
reductively methylated according to the general procedures
described by Pon et al. dialysed
21
measured by the method of
1 A280 of IgGs corresponds to 840 pg of protein.
Reductive methylation IgGs
determined separately for
was
was
About 3A
units of Batch D IgGs
period of 2 hours with 3 changes of buffer. 14C-Formaldehyde (aqueous solution)
was
Reactions
sodium borohydride
were
a
final
Two 204l aliquots of
concen-
a
0.5%
then added with 1 minute intervals, and the reduction
carried out for 15 minutes at 00.
Samples
were
then dialysed against IgG
buffer overnight until all unreacted formaldehyde activity of labelled IgGs
was
removed.
was
Specific
449 dpm/ig protein.
Incubation of ribosomes and subunits with IgGs and general procedures of Zamir et al.
The
a
carried out at 00.
were
added to the IgGs to
tration of 3mM and incubated for 30 seconds.
tion.
fast
were
280
against 50mM sodium borate buffer pH 9 containing 200mM KC1 for
30S subunits (200-300 A 260units/mi)
were were
sucrose
followed for heat activa-
mixed with
of buffer C, and incubated at 400 for 30 minutes.
gradient analysis
an
equal volume
Activated subunits
were
chilled on ice, and immediately used for IgG interaction.
Activated
or
unactivated 30S subunits
were
incubated with the IgGs
indicated in the figure legends in buffer D at 37° for 15 minutes. end of incubation, the 1004l samples were chilled
layered in
an
over
10-30%
sucrose
gradients in buffer E.
SW 50.1 rotor at 32,000
rpm
Radioactivity
was
were
At the
ice, and immediately Gradients
for 4.5 hours at 4
dripped manually, 3-4 drop fractions at 260 nm.
on
as
.
were
Gradients
centrifuged were
collected, and absorbance measured
measured by liquid scintillation counting in
Triton X-100 cocktail. For the dissociation-reassociation experiments, 70S ribosome couples were
incubated at 370
chilled
on
1M Mg
acetate.
Gradients
analysed
as
indicated, in buffer F.
ice and immediately brought were
as
Samples
were
centrifuged in
analysed an
up
After incubation, samples
were
to 15mM Mg
on sucrose
concentration by adding gradients in buffer G.
SW 50.1 rotor at 32,000 rpm for 3 hours, and
described above.
Cross-linking of 30S subunits 30S subunits were fast dialysed against a buffer containing 10mM Trisacetate for a period of 1.5 hours with 5 HC1 pH 7.8, 50mM KC1, and lmM Mg
changes of 300 ml each.
808
Dialysed 30S subunits freed of mercaptoethanol were
Nucleic Acids Research incubated with a cross-linking control reagent for 30 minutes at 300. PM and o-PDM were freshly dissolved in acetone while p-PDM was dissolved in dimethyl formamide (due to low solubility in acetone) at a concentration of lOmM. The reagents were added to the 30S subunits at a final concentration of 0.4 mM exactly as described by Chang and Flaks.23 Under our conditions, the ratio of subunits to reagent was 1:30. After a 30 minute reaction, a-mercaptoethanol was added to a final concentration of lOmM, and incubations continued for 10 minutes. Samples were dialysed against buffer H. Gel electrophoresis
Cross-linked proteins were analysed by polyacrylamide gel electrophoresis at pH 4.5 as described by Hardy et al. 4 30S proteins were extracted by the RNase method of Nashimoto et al. 25 Two different gel systems were used for SDS polyacrylamide gel electrophoresis. 10% gels were used in a Weber's system, 6 and 13.5% gels were employed in a Laemmli system. 27 Samples for electrophoresis were prepared by heating 30S subunits at 650 for 10 minutes in 1% SDS, 2% B-mercaptoethanol, 4mM EDTA and 4M Urea. Gels were scanned by
ISCO gel scanner model 659, and absorbance recorded by ISCO Model UA-5 absorbance monitor.
Binding
of
C-labelled IF3 to 30S 14C-labelled purified initiation factor IF3 was a generous gift from Drs. Barry Cooperman and Marianne Grunberg-Manago. 30S subunits were incubated with the labelled IF3 in buffer E at 370 for 15 minutes. ° Incubated samples were layered on sucrose gradients in the incubation buffer and analysed as described for 30S-IgG dimers except that the samples were processed directly for scintillation counting unless otherwise stated. tRNA binding assays
R17 RNA dependent fMet-tRNA binding to 30S subunits was done according R17 RNA was isolated from R17 phage grown
to general published procedures.
according to a procedure described by Dr. R. F. Gestland (personal communication). 0.73 A260 units of MRE 600 30S subunits (48 pmoles) were incubated with 0.44 A 260 units of R17 RNA, 0.38 A 3H fMet-tRNA (19.8 260 units of pmoles; specific activity 11,005 dpm/pmole), optimized amounts of ammonium sulfate fractionated crude initiation factors, 28 and 1.6 mM GTP. tRNA binding reactions were carried out in a 5Ol volume of buffer J, at 370 for 15 minutes. At the end of incubation samples were chilled on ice, and diluted with 1.5 ml of cold buffer J. Samples were filtered on Millipore Type HA filters followed by 3 washes with the same buffer.
Dried filters
were
counted in 809
Nucleic Acids Research PPO-POPOP scintillation fluid.
The counting efficiency was 21%.
Poly U dependent Phe-tRNA binding to 30S subunits was carried out
according to a procedure similar to that described above. 0.74 A260 units of 30S subunits (49 pmoles) were incubated with 0.84 A260 units of Poly U, and 1 A 260 unit of C Phe-tRNA (38 pmoles; specific activity 461 dpm/pmole) 30 at 0° for 60 minutes. Reactions were in 100 4l of 10 mM Tris-HCl buffer
(pH 7.2) containing 60 mM NH 4 Cl, 10 mM Mg acetate, and 6 mM B-mercaptoethanol. tRNA bound to 30S subunits was determined as described above. The counting efficiency was 74%. tRNAs were charged using procedures kindly provided by Dr. A. E. Johnson. RESULTS Interaction of antibody with 30S particles Sucrose gradient centrifugation was used to study the interaction of
m6A
antibodies with 30S subunits isolated from E. coli strain MRE 600. Fig. la shows a typical yield of 30S dimer formation upon incubation of 30S subunits with the IgGs. The control experiment shown in Fig. lb, demonstrates that the methyl deficient 30S particles from kasugamycin resistant strain TPR 201, fail to form dimers; only free 30S particles and IgGs are found after incubation. No dimers are seen with 30S subunits that have been incubated under similar conditions in the absence of IgGs. We have also failed to observe dimers upon incubation of 30S with non-immune IgGs (data not shown). These experiments demonstrate the specificity of the antibody reaction resulting in dimer formation. m6A Although the antibodies recognize the m6A 2 2 sequence in the isolated 30S subunits as shown in Fig. la, they do so only with the inactive conformation of 30S. Zamirand coworkers observed that heat activation of 30S subunits characteristically enhances the binding of 50S subunits and binding of transfer RNA in the presence of messenger RNA. ' In our hands, heat activation produced a 2-3 fold stimulation in R17 RNA dependent fMet-tRNA binding to 30S subunits and a 24 fold stimulation in Poly U dependent Phe-tRNA binding to 30S subunits. See Table I. Incubation of IgGs with 30S subunits yields no heat activated according to the procedures of Zamir et al., is rendered inaccessidimers (Fig. lc). Apparently, the methylated sequence ble for dimer formation upon heat activation of 30S subunits. To demonstrate that direct interaction of IgGs with the subunits is required for dimer formation, we employed radioactive IgGs prepared by reductive methylation with 14C-formaldehyde as described in materials and methods. 810
Data
presented in Fig. 2 demonstrate that dimer formation involves
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