Volume 12 Number 3 1984
Nucleic Acids Research
Binding of tRNA alters the chemical accessibility of nucfeotides within the large ribosomal RNAs of
E. coli ribosomes
N.Meier and R.Wagner
Max-Planck-Institut fUr Molekulare Genetik, Abteilung Wittmann, Ihnestrasse 63-73,
1000 Berlin-Dahlem, FRG
Received 20 October 1983; Revised and Accepted 15 December 1983
SUMMARY
Functionally active 70S ribosomes were chemically modified
with dimethylsulfate (DMS) in the presence and absence of bound
tRNA. The ribosomal 16S RNA and 23S RNA were extracted, separated and labeled radioactively at their 3'-ends. DMS modification
sites within the last 200 nucleotides from the 3'-ends were investigated on sequencing gels, after borohydride reduction and
aniline catalyzed strand scission of the isolated RNA's.
tRNA binding caused enhanced reactivity at 9 nucleotide
positions while three sites showed decreased reactivity in the
16S RNA. The effects of bound tRNA on the modification of 23S
RNA were limited. Only one enhancement was observed in the presence of bound tRNA. mRNA binding alone showed two more sites
with enhanced reactivity, however. The results are consistent
with the view that the sequence 1400-1500 of the 16S RNA plays
an important functional role in the translating ribosome and
possibly constitutes part of the tRNA binding site.
INTRODUCTION
Studies on the primary and secondary structures of the ribosomal RNA's have made tremendous progress in the last few years.
Reasonable secondary structural models based on experimental as
well as theoretical evidences have been proposed (1-4) and information on the tertiary folding is emerging gradually (5).
The functional importance of the ribosomal RNA molecules
had long been neglected but numerous results confirm today's
view of them as dynamic molecules which are directly implicated
in a number of the catalytic functions of the translating ribosome (6-8) .
The presence of rRNA within catalytic centers of the ribosome has been demonstrated by affinity labeling and cross-linking
experiments (9-11). Furthermore, clear evidence exists for a
direct participation of the 3'-end of the ribosomal 16S RNA in
© IRL Press Limited, Oxford, England.
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the recognition of natural mRNA initiation sites (12,13).
We have focused our study on the effects of bound tRNA on
the large rRNA's of the 70S ribosome by comparing the dimethylsulfate modification of accessible nucleotides within the ribosomal RNA's. For technical reasons only about 200 nucleotides
from the 3'-end were monitored. The results are discussed with
respect to the known DMS modification sites in the free RNA
molecules (14) and with other known topographical data on the
tRNA binding sites.
MATERIALS AND METHODS
Phe
t R N A y e a 3 t and poly(U) were obtained from Boehringer, Mannheim. [32p]pcp (specific activity 2000-3000 Ci/mmole) was bought
from Amersham/Buchler, Braunschweig. Dimethylsulfate and Na borohydride were from Merck, Darmstadt. Diethylpyrocarbonate was
from Eastman, Kodak, and hydrazin and aniline were from Aldrich.
Acrylamide, N',N'-methylene-bisacrylamide and N,N,N',N'-tetramethylenediamine were obtained from Bio Rad, Richmond. Polynucleotide ligase (E.C. 6.5.1.3) was a product of PL Biochemicals,
Milwaukee. X-ray films Medical were from Fuji, Japan. 70S ribosomes were prepared as described (15).
tRNA binding
Phe
Binding of tRNA
was performed by incubating 100 pmoles
70S ribosomes together with 40 ug poly(U) and 200 pmoles uncharged tRNA in a total volume of 100 til 50 mM Na cacodylate, pH 7.2,
150 mM KC1, 20 mM MgCl,, 1.5 mM DTE and 2 mM EDTA for 15 min at
0°C.
Modification reaction
To 100 ill of 70S ribosomes or 70S ribosome ^tRNA^inRNA
complexes (100 pmoles each) 2 ill of a fresh mixture of DMS in
methanol (1:4 v:v) was added. The final DMS concentration was
50 mM. Samples were incubated for 15 min at 37°C. The reaction
was terminated by rapidly mixing the samples with 1 volume of
cold ethanol (-80°C).
Isolation of ribosomal RNA's
After the ethanol precipitation the modified ribosomes
or the unmodified controls were redissolved in 200 \il 50 mM
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Tris borate, pH 7.2, 150 mM NaCl, 15 mM Na citrate, 10 mM EDTA
and 1% (w/v) SDS. The samples were three times extracted with
an equal volume of phenol saturated in the same buffer. Residual
phenol was removed from the water layers by two ether extractions. The ribosomal RNA's were precipitated with 2.5 volumes of
ethanol at -20°C for 2 hours. The RNA was dissolved in 50 mM
Na acetate pH 6.0, 20 mM Na borate and separated on a linear 10%
to 30% sucrose gradient using a SW40 rotor (35.000 rpm, 4°C,
14 hours). 16S and 23S RNA peaks were collected separately and
precipitated with 2.5 volumes of ethanol for 4 hours at -20°C.
Samples were dissolved in 100 mM NH. acetate pH 6.0, 20 mM Na
borate and reprecipitated with ethanol.
3'-end labeling of ribosomal RNA's
rRNA's were 3'-end labeled using 50 nCi [ P]pCp and RNA
ligase as described by Bruce and Uhlenbeck (16).
Chain scission reaction
RNA chains were cleaved at methylated guanosine positions
by the method described by Peattie (17). Fragments were separated on 12% polyacrylamide, 8 M urea gels (40 x 35 x 0.04 cm) at
constant 25 Watt until the xylene cyanol blue marker dye had
migrated about 15 cm (3-4 hours). RNA fragments were made visible
by autoradiography.
Identification of methylation sites
Bands of interest were cut out according to the autoradiograms and the RNA fragments were extracted as described (18).
Unambiguous assignment of the 5'-terminal nucleotide (the DMS
modified position at which cleavage occurred) was achieved by
chemical sequencing of the isolated fragment using the method
described by Peattie (17).
Quantitation of band intensities
Differences in the intensities of the gel bands were sometimes difficult to assess precisely because of the lack of a
constant reference band. Only those effects resulting in a
clearly visible intensity difference between different tracks of
the same gel were therefore regarded as significant. To obtain
a more accurate intensity estimation the autoradiograms were
scanned using a Vitatron TLD100 densitometer.
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RESULTS
Stability of modified complexes
Ph<=
Binding of tRNA
to the 70S ribosomes and the specificity
of the binding was tested as described in (18) . Reaction conditions employing 50 mM DMS and incubation at 37°C for 15 min
were found to be optimal. They gave sufficient modification rates
on the ribosomal RNA's and were mild enough not to distort the
ribosoraal structure and function. This could be demonstrated by
comparison of the tRNA binding activity before and after DMS
modification. The DMS modification did not reduce the tRNA binding activity by more than 12%.
Separation of rRNS's
Ribosomal RNA's from modified 70S ribosomes and 70S ribosome'^tRNA'vmRNS complexes were extracted with phenol and separated on linear sucrose gradients. The RNA was tested before and
after the modification reaction on composite agarose acrylamide
gels (Fig. 1,a and b ) . No difference in the electrophoretic
mobility can be detected. Hence it was concluded that the DMS
modification of the ribosomes does not fractionate or damage the
ribosomal RNS's to any detectable extent. Aliquots from the
pooled gradient fractions containing 23S RNA and 16S RNA were
analysed for purity and shown to be homogeneous on the same gel
system (Fig. 1 ,c-f) .
Effects of tRNA- and mRNA binding on the modification of the
ribosomal RNA's
Ribosomal RNA extracted from ribosomes or ribosome'vtRNA'^
mRNA complexes after modification and separation were radioactively labeled at their 3'-ends. The RNA chains were cleaved at the
modified positions by borohydride reduction of the DMS modified
guanosines followed by an analine catalyzed strand scission
exactly as described (17). The resulting fragments were separated
on 12% sequencing gels in the presence of 8 M urea alongside
untreated RNA controls. As additional controls every RNA sample
was separated with and without borohydride and aniline treatment.
Samples that had not been treated with borohydride and aniline
are indicated by "'". DMS modification followed by aniline catalyzed strand scission resulted in a specific fragmentation of
the ribosomal 16S (Fig. 2, lanes b,c,d) and 23S RNA's (Fig. 3,
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a bc d e f
— 23 S
— US
BPB
Fig. 1 : Separation of ribosomal RNA's on acrylamide-agarose gels
Gels were prepared and samples are separated as described in
(15)
a) total RNA without DMS modification.
b) total RNA after DMS modification
c) 23S RNA isolated after gradient separation without
DMS modification.
d) 23S RNA isolated after gradient separation after
DMS modification.
e) 16S RNA isolated after gradient separation without
DMS modification.
f) 16S RNA isolated after gradient separation after
DMS modification.
lanes b,c,d). However, a number of bands show up in the untreated
control RNA's (Fig. 2 and 3, lanes a) and have to be considered
when different samples are to be compared. The borohydride and
aniline treatment had no effect on the fractionation of the
control RNA (compare lanes a) and a') in Figs. 2 and 3 ) .
Comparison of tracks, b,c) and d) reveals intensity differences for a number of bands in Figs. 2 and 3. A reduction in
the band intensity is an indication for a reduced DMS reactivity
at that particular nucleotide, whereas an enhancement of the intensity of a gel band reveals an increased DMS reaction.
In Fig. 2 where 16S RNA samples are separated, 12 clear
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,
,
f
b b C C d d
_
—
*"*~ mm
Fig. 2: tRNA dependent DMS
modification of 16S RNA
Gel separation of fragments
obtained after chain scission at
modified nucleotides of 16S RNA
a) 70S t.c. unmodified, b) 70S
t.c. modified with DMS,
c) 70SM:RNA complex modified with
DMS, d) 70S^tRNA^mRNA complex
modified with DMS. Samples labeled
"•" are not aniline treated. They
are only included to show that in
the absence of the aniline reaction
some intermediate products are
formed as a consequence of the DMS
reaction. In some cases the
fragmentation with and without
aniline treatment are identical.
Numbers indicate fragments with
intensity differences. XC denotes
the xylene cyanol marker dye.
b
_
11
12
XC
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i
3 3
mm
.
,
,
b b C C d d b
,^
.. ^ ^
Fig. 3: tRNA and mRNA dependent
fragmentation of 23S RNA
Gel separation of fragments obtained
after chain scission at modified
nucleotides of 23S RNA. Arrangement
of samples is the same is given
for Fig. 2.
a
p*
xc
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Fig. 4: mRNA dependent fragmentation
o f 2 3 S RNA
An enlarged section of a similar
separation as shown in Fig. 3 is given.
In e) an additional sample is separated
where 70S ribosomes were modified with
DMS in the presence of mRNA but in the
absence of tRNA.
intensity differences can be detected when lanes b) and d) are
compared. In b) samples are separated that had been extracted
from ribosomes modified with DMS in the absence of tRNA and mRNA.
This track is therefore the main reference track and for a better
comparison it is shown twice in Figs. 2 and 3.
In c) RNA samples are separated that had been modified in
the presence of tRNA but in the absence of poly(U). Lane d)
shows the separation of RNA extracted from ribosomes which had
been modified in the presence of tRNA and poly(U).
Generally the protection or enhancement effects visible in
Fig. 1 for the 16S RNA samples are apparent when the modification
was performed in the presence of tRNA but in the absence of
poly(U) (compare lanes b) and c)). They are more pronounced,
however, when the tRNA was bound in the presence of poly(U).
The intensity differences visible for bands 7 and 8 were
also detected when the samples were not reacted with borohydride and aniline (lane d ' ) . They are, however, absolutely
reproducible and strictly dependent on whether the DMS modification was performed in the presence or absence of tRNA and
mRMA.
A somewhat different situation exists for the effects of
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FRAGMENTS FROM
23SRNA
16SRNA
R YRY
2834 —
XC —
RY
- f
Chemical
sequencing of RNA bands
extracted from gels shown
in Fig. 2 and 3
Two 23S and one 16S RNA
examples are shown. A
pyrimidine (Y; U>C) and a
purine (R; A>G) track is
shown for each fragment.
Fragments are extracted
from the gels shown in
Fig. 2 and 3.
—xc
bound tRNA and mRNA on the methylation of about the last 200
nucleotides of the 23S RNA. The arrangement of lanes in Fig. 3
for the separation of 23S RNA samples is the same as shown in
Fig. 2 for the 16S RNA samples. Only one intensity difference
was apparent for the 23S RNA when the ribosomes were modified in
the presence and absence of tRNA and mRNA (band 1, Fig. 3 ) . The
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c
o_
Table 1:
Localisation of tRNA/mRNA induced effects on the modification of 16s and 23S RNA
CD
>
tRNA induced effects in 16S RNA
NO. of
gel bands
1
Position
tRNA induced
effects
P
G
1401
2
E
G
3
E
G
1405
1415
4
E
G
1442
5
6
E
E
G
1453-55
G
7
P
U
8
9
10
11
12
E
E
E
E
P
C
tRNA and mRNA induced effects in 23S RNA
No. of
gel bands
1
Effects induced
by
tRNA
mRNA
Position
E
A
A
2
E
A
3
E
A
E
3)
2826
2835
2855
1457-59
1463/64 2»
1466/67 2 )
G
1473
1475
G
1486
G
1497
G
1)Strongest tRNA induced protection
2(Aniline independent
3(Effect occurs in the presence of tRNA and mRNA alone, but is markedly enhanced when both
are present.
P denotes protected, E enhanced reactivity for DMS modification,
o
a.
e
CD
O)
O
3"
Nucleic Acids Research
effect can be seen more clearly in Fig. 4 where a longer separation of an experiment identical to that shown in Fig. 3, is
presented. In addition in lane e ) , Fig. 4, the RNA investigated
came from ribosomes that had been modified in the presence of
poly(U) but in the absence of tRNA. Two more intensity differences became apparent (bands 2 and 3, Fig. 4) which are strictly
mRNA dependent and can no longer be detected when both tRNA and
raRNA are bound to the ribosome (lane d, Fig. 4 ) . Although this
experiment was only performed once it is a good indication for a
mRNA (poly(U)) induced change in the chemical accessibility of
the corresponding nucleotides. It is interesting to note that
all the effects detected in the different 23S RNA fractions are
aniline independent as were those observed for band 7 and 8
of the 16S RNA samples.
Bands that showed intensity differences were cut out from
the sequencing gels and the 5'-nucleotide was determined by chemical sequencing. An example is shown in Fig. 5. For the unambiguous identification of the methylated nucleotide only a C- and a
A-reaction had to be performed with the fragments to be analysed.
All the fragments labeled in Fig. 2 and 3 and several control
fragments not labeled were sequenced to determine the 5'-nucleotides. In all cases the sequences of the fragments determined
were in accordance with the sequences expected from the 3'-ends
of 16S and 23S RNA. This indicates that no fragments originating
from some other area of the large RNA's were investigated.
Table I summarizes all the effects detected in 16S and 23S RNA
and gives the nucleotide positions to which the effects were
localized. All the effects were reproduced at least 3 times with
identical results with the exception of the experiment shown in
Fig. 4, lane e ) , which was only performed once.
Additional intensity differences can be seen in Fig. 2
above band 1, which were not labeled. The corresponding fragments have so far not been exactly identified because of their
size. Some other differences were not reproducible and usually
showed cuts at the corresponding control tracks. They were
therefore not considered as significant.
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DISCUSSION
The structural information gained from chemical modification studies of a complex system like the ribosome, is limited
to conditions where the particles investigated do not denature
but remain intact and active. Using DMS as a modifying agent
we and others (19-21) have shown that under the modification
conditions used in this study there is no significant loss of
activity of the ribosomes, and no change in the structure of
the particles investigated could be detected.
However, during the study a number of problems intrinsic to
the system were encountered. Several cuts were present in the
ribosomal RNA's independent from the modification and work up
procedure (Figs. 2 and 3, lane a ) . These cuts, however, are
very reproducible and are present in the ribosomal preparation
and/or are introduced during the 3'-ligase reaction. The concentration of these artificial fragments is greatly overestimated from the intensity of the bands on the sequencing gels, because these smaller fragments are much more effectively ligated
to the [ P]pCp by the ligase. If gels were stained with toluidine blue only the unfractionated molecules could be detected.
Care was taken whenever a cut appeared in the control and the
only effects considered as significant were those where no or
only very weak cuts were visible in the RNA control tracks.
A second problem arose when the intensities of bands on the
sequencing gels had to be estimated. Although equal amounts of
radioactive material were applied to the sequencing gels, the
intensity of the starting material (intact rRNA's) varied to
some extent as a result of different yields during the ligase
reaction. The presence of free [ P]pCp, which could not always
be removed quantitatively often made impossible a direct comparison of corresponding bands. For a more accurate estimation of
the band intensities the relative intensities had to be compared.
They were obtained by calibration with bands of constant intensity.
The method used is further limited by the fact that only
guanosines will normally be detected when DMS is used as a modifying agent. If other nucleotides are to be monitored then DEP
or hydrazine has to be used. In some cases (bands 7 and 8, Fig. 2,
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and bands 1-3, Fig. 4) differences in band intensities between
the untreated controls and the RNA from ribosomes modified in
the absence of tRNA and mRNA were detected, without the borohydride and aniline treatment. We can not explain the mechanism
of this cleavage process. The effects are highly reproducible,
however, and never appear in control ribosomes. We feel therefore that these effects are specifically induced in the complexed particles by some unknown reaction of the DMS with parts of
the RNA that only react favourably in the presence of tRNA. These
aniline and borohydride independent cuts do not occur at guanosines but at uracyl, cytosine and adenosine positions.
Only about 200 nucleotides from the 3'-ends of the RNA molecules are analyzed in this study. The analysis of the 5'-end and
the central regions of the large rRNA's will be considerably more
difficult. Good evidence exists, however, for the participation
of the 3'-terminal sequences of the rRNA in the functions investigated. This is confirmed, at least for 16S RNA, by our
results.
Protection from or enhancement of the chemical modification,
as observed in this study, can either be interpreted as direct
shielding by the bound substrate or by a change in the chemical
environment, that could also be transmitted from somewhere else
in the molecule. We are not able to distinguish between these
two possibilities. In some cases, however, where for example
direct cross-linking evidence demonstrates the close proximity
of bound tRNA and C.. o o of the 16S RNA a direct shielding effect
seems very likely to explain the protection of G.^Q^.
If the sites of DMS modification are placed in one of the
secondary structural maps as proposed by Zwieb (1), Noller (2)
or Stiegler (3) no clear preference for the modification of
single- or double stranded RNA regions can be detected.
This is in accordance with the known specificity of DMS
which attacks the N-7 position of purines and can therefore react
with single- and double stranded RNA molecules. Hence, no clear
conclusions with respect to the different secondary structural
models proposed can be drawn. DMS is, however, a very suitable
probe to test tertiary interactions and its value has been demonstrated in exploring the structure of the 3'-terminal domains
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of three different ribosomal RNA's (14). There is generally a
good correlation between the DMS modification sites found in the
free 16S RNA and this study. Some noticeable exceptions exist,
however. The guanosine positions G 1 4 Q 1 and G i457_9 a r e protected
from DMS modification in the free RNA (14) but are accessible in
the ribosome. One has to conclude that there are local structural
changes between the free 16S RNA and the RNA within the ribosome.
Note that the modification of the free RNA was performed with 8
times higher DMS concentrations and for longer reaction times.
Effects of tRNA bound to the ribosomal P-site have been investigated before using tritium exchange and chemical modification studies (21,22) . in both cases where the structure of the
tRNA was investigated the observed effects were independent of
the presence of mRNA. These findings are partly confirmed in our
study where effects on the ribosomal RNA's by bound tRNA were observed even in the absence of mRNA (see Figs. 2 and 3, lances c)
and d ) .
It is interesting to note, that tRNA binding has a strong
effect on multiple sites within the 3'-end domain of the 16S RNA,
whereas only a limited number of effects can be detected within
the last 200 nucleotides of the 3'-end of the 23S RNA. This finding agrees well with the view that the tRNA binding and decoding
domain is composed partly of 16s RNA (9,10) and partly of the
ribosomal subunit interface (23-25). The corresponding part of
the interface of the large ribosomal subunit seems to be mainly
composed of proteins (15).
Similar results with respect to the involvement of 16S and
2 3S RNA in the translation mechanism have been reported by Brow
and Noller (8), where the chemical modification of free ribosomes and polysomes were compared.
Differences in the DMS modification of free subunits and
reassociated 70S ribosomes support the findings that the 16S
and 2 3S RNA's contribute differently to the interface domains
of the two subunits. These results will be reported elsewhere.
ACKNOWLEDGEMENT
We like to thank Prof. H. G. Wittmann for his support,
K. Ashman for carefully reading the manuscript, R. Brimacombe
for providing us with unpublished information on RNA secondary
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structure. The helpful discussion of S. Marlow is greatly
appreciated. The work was supported by the Deutsche Forschungsgemeinschaft, SFB9.
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