Effects of antisense DNA against the
[alpha]-sarcin stem-loop structure of the ribosomal 23S rRNA
Effects of antisense DNA against the [alpha]-sarcin stem-loop structure of the ribosomal 23S rRNA
Hellmuth-Alexander
Meyer
1
,
Francisco
Triana-Alonso
1
,
Christian M. T.
Spahn
1
,
Tomasz
Twardowski
1,2
,
Andrzej
Sobkiewicz
1,2
and
Knud H.
Nierhaus
1,
*
1
Max-Planck-Institut für Molekulare Genetik, AG Ribosomen, Ihnestra[beta]e 73, D-14195
Berlin
,
Germany
and
2
Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12,
61-704
Poznan
,
Poland
Received July 1, 1996;
Revised and Accepted September 2, 1996
ABSTRACT
Antisense DNAs complementary against various sequences of the
[alpha]
-sarcin domain (C2646-G2674) of 23S rRNA from
Escherichia coli
were hybridized to naked 23S rRNA as well as to 70S ribosomes. Saturation
levels of up to 0.4 per 70S ribosome were found, the identical fraction was
susceptible to the attack of the RNase
[alpha]
-sarcin. The hybridization was specific as demonstrated with RNase H
digestion, sequencing the resulting fragments and blockage of the action of
[alpha]
-sarcin. The RNase
[alpha]
-sarcin seems to approach its cleavage site from the 3
'
half of the loop of the
[alpha]
-sarcin domain. Hybridization is efficiently achieved at 37
o
C and can extend at least into the 3
'
strand of the stem of the
[alpha]
-sarcin domain. However, the inhibition of
[alpha]
-sarcin activity is observed at 30
o
C but not at 37
o
C. For a significant inhibition of poly(Phe) synthesis the temperature had to be
lowered to 25
o
C. The results imply that the
[alpha]
-sarcin domain changes its conformation during protein synthesis and that
the conformational changes may include a melting of the stem of the
[alpha]-sarcin domain.
INTRODUCTION
Hybridization of antisense DNA to exposed regions of rRNAs in intact ribosomes
and ribosomal subunits has been introduced by Bogdanov and co-workers (
1
,
2
) and has been used to analyze structure and function of ribosomes. The effects
of specific hybridization on the ribosomal functions has been studied by Hill
and co-workers (for review see ref.
3
) using an impressive set of antisense DNAs. The results with oligo(DNA)
complementary to sequences of the 16S rRNA indicated, for example, that the
region containing the bases 518-525 might be part of the mRNA binding site, either poly(U) and tRNA
Phe
competed with probes crossing C1400, whereas only the presence of both mRNA and
cognate tRNA blocked probes against the bases 1534-1541 which contains the anti-Shine-Dalgarno region. Probes against the bases 807-809 blocked tRNA binding, but tRNA lacking the 3 or 4
3'-terminal nucleotides (nt)could be accommodated. tRNA could also
effectively compete with probes complementary to bases 2497-2505 at the peptidyltransferase ring of the 23S rRNA. Others have linked
immuno-specific ligands or avidin to antisense DNA and identified the
hybridization site by electron microscopic methods (
4
,
5
). Attachment of psoralen (
6
) or photoaffinity labels (
7
) enabled site-specific crosslinks. The latter technique was particularly successful in
identifying specific neighborhoods. For example, a photo-sensitive affinity label attached to an antisense DNA complementary to
bases 2497-2505 of the peptidyltransferase ring of the 23S rRNA showed a specific
crosslink to the ribosomal protein L3 (
8
), the label of a probe against bases 1397-1405 of 16S rRNA at or near the mRNA decoding site was specifically
incorporated into proteins S1, S7, S18 and S21 (
7
). These few examples illustrate that hybridization of antisense DNA
complementary to sequences of rRNAs represent an important complement and
extension of the arsenal of methods assessing the quaternary structure and its
relation to important functional domains of the ribosome.
The [alpha]-sarcin stem-loop structure of the 23S-type rRNA contains in its loop the longest universally
conserved sequence (12 nt) of all rRNAs. The structure and function has been
intensively studied by Endo and Wool (
9
, for review see ref.
10
). The hydrolysis of one phospho-diester bond after G2661 (
Escherichia coli
numbering) by the highly specific RNase [alpha]-sarcin blocks all elongation factor dependent reactions but does
not affect factor independent ribosomal functions (
11
). Binding of EF-G in the presence of fusidic acid completely prevents cleavage of the
RNase [alpha]-sarcin (
12
), and binding of either elongation factor protects this structure against the
attack of modifying reagents (
13
). Hybridization of an oligo(DNA) to the loop of this structure was first
thought to have an disastrous effect on the integrity of the 50S subunit (
14
), but this dramatic effect could be traced back to the harsh incubation
conditions and had nothing to do with the specific probe (
15
). The authors of ref.
14
asserted that the entire region of the [alpha]-sarcin domain of 23S rRNA was practically unavailable to probe
binding although a 14mer covering the sequence 2654-2667 could trigger a specific cleavage of RNase H. These observations
contrasted with a report suggesting that the microinjection of an antisense DNA
complementary to the [alpha]-sarcin loop of 28S rRNA abolishes protein synthesis in
Xenopus
oocytes (
16
) as well with some preliminary results which were obtained with some probes
against this region and according to which hybridization had severe
consequences for function and structure of ribosomes (
17
); one of us seemed to reproduce this effect with plant ribosomes (
18
). In order to clarify the controversial situation we re-evaluated the effects of antisense DNA covering the [alpha]-sarcin stem-loop structure of 23S rRNA. Here we describe the
results of a study analyzing ribosomal functions
in vitro
in the presence and absence of the corresponding oligonucleotides.
MATERIALS AND METHODS
Preparation of tightly-coupled 70S ribosomes and subunits
Tightly-coupled 70S ribosomes as well as 50S subunits were prepared from
E.coli
AB301, strain D10 as described (
19
). NH
4
Cl washed 70S ribosomes were obtained by resuspending and incubating the crude
70S pellet with 1 M NH
4
Cl, 10 mM HEPES, pH 7.5 (0oC), 6 mM [beta]-mercaptoethanol for 5 h at 0oC before zonal centrifugation under the conditions of the
`tightly coupled ribosomes' (
19
).
Preparation of DNA oligonucleotides
The DNA oligomers (listed in Table
1
) were obtained from TIB Molbiol, Berlin. Probes were purified after
detritilation by standard 20% (w/v) sequencing polyacrylamide gel electrophoresis (
20
).
Table 1
.
The antisense oligo(DNA)s used in this study were aligned to the [alpha]-sarcin domain of the 23S rRNA
Purified DNA probes were 5"-end-labeled using T4 polynucleotide kinase and [[gamma]-
32
P]ATP as described by Maxam and Gilbert (
20
) with the omission of the dephosphorylation step. The specific activities of
the labeled probes were in the range of 700-1000 c.p.m./pmol. The same procedure was used for `cold' phosphorylated
probes.
Hybridization assays
Hybridization of the various DNA oligomers to ribosomes were performed in the
ionic conditions of the poly(Phe)-synthesis system described by Bartetzko and Nierhaus (
21
), 20 mM HEPES-KOH, pH 7.6 (0oC), 3 mM MgCl
2
, 150 mM NH
4
Cl, 2 mM spermidine, 0.05 mM spermine and 4 mM [beta]-mercaptoethanol. The DNA oligonucleotides were denatured by heating
for 2 min at 70oC. After cooling on ice 10 pmol of 23S rRNA or 70S ribosomes were
immediately mixed with the antisense DNA at a 35-fold molar excess if not otherwise indicated in a volume of 10 [mu]l and incubated for 10 min at 37oC followed by an incubation at 2 h at 0oC. The samples were diluted with 2 ml of the same ionic
concentration and filtered through nitrocellulose filters. In order to reduce
the background binding of the radioactive oligo(DNA)s the filters were soaked
in the same buffer, degassed and then transferred to the same buffer containing
2 mg/ml of each poly(U) and poly(C) before use.
RNase H and
[alpha]
-sarcin assays
[alpha]-sarcin and RNase H were generous gifts of Dr N. Ulbricht (FU
Berlin) and Dr R. Brimacombe (MPI Berlin), respectively. The digestion of 50S
subunits, 70S ribosomes or 23S rRNA with [alpha]-sarcin or RNase H in the presence or absence of antisense DNA were
performed under the ionic conditions of the hybridization reaction described
before.
RNase H was used to demonstrate the specific binding of the DNA-oligomers to 50S subunits, 70S ribosome and naked 23S rRNA. The reaction
mixture contained 10 pmol of 70S ribosomes or 23S rRNA, an excess of DNA
oligomers as indicated. After the hybridization incubations as described above
the digestion reaction was started by adding 5 [mu]l containing 5 [mu]g RNase H in the same buffer, an incubation of 10 min at 30oC followed. Eight microlitres were mixed with 8 [mu]l of stop-buffer [50 mM Tris-HCl pH 7.5 (0oC), 1 mM EDTA, 8 M urea, 0,05% (w/v) xylene
cyanol, 0.05% (w/v) bromophenol blue and 1% (w/v) SDS] and heated for 3 min at
80oC. The RNA digestion products were analyzed by polyacrylamide gel electrophoreses [4 or 8% polyacrylamide, 7.5 M urea 0.1% (w/v) SDS, 90 mM Tris-borate pH 8.0 (0oC) and 2.5 mM EDTA] and the RNA bands were visualized by
staining with ethidium bromide. The RNA bands were quantified by densitometry.
The relative amount of the specific [alpha]-sarcin RNA-fragment was normalized to the 5S band in the same lane.
The mixture for the [alpha]-sarcin digestion after the hybridization incubations contained 24
pmol 70S, 840 pmol DNA oligomer and 1.2 [mu]g [alpha]-sarcin in 40 [mu]l. During an incubation at 30oC, 5 [mu]l samples were taken out at various time intervals
and were immediately mixed with 5 [mu]l stop-buffer and subjected to gel electrophoresis as described above.
Poly(Phe)-synthesis
tRNA
Phe
was obtained from Biogenes, Berlin, poly(U) and GTP was purchased from Boehringer Mannheim. Long poly(U) strands were isolated by sucrose-gradient centrifugation. Twenty-five milligrams of commercial poly(U) was dissolved in 1.5 ml H
2
O and applied on a sucrose gradient [5-20% (w/v) in 20 mM HEPES-KOH (pH 7.6) at 0oC], 500 mM NaCl and 0.1 mM EDTA and centrifuged for 30 h at 25 000
r.p.m. in a SW 27 rotor (Beckman). The fractions containing the fast migrating
poly(U) material were collected and poly(U) isolated by ethanol precipitation.
The acylation and acetylation of tRNA
Phe
were done as previously described (
22
), the purification of the charged tRNA followed Geigenmüller and Nierhaus (
23
). The specific activity of [
3
H]Phe-tRNA was ~15-20 c.p.m./pmol, that of Ac[
14
C]Phe-tRNA 1000 c.p.m./pmol.
The poly(Phe)-synthesis followed Bartetzko and Nierhaus (
21
) except that the volumes of the binding reaction and of the synthesis mixture
were reduced to 50 and 30 [mu]l, respectively (total volume 80 [mu]l). The ionic conditions of the system were 20 mM HEPES-KOH, pH 7.6 (0oC), 3 mM MgCl
2
, 150 mM NH
4
Cl, 2 mM spermidine, 0.05 mM spermine and 4 mM [beta]-mercaptoethanol. One aliquot contained 10 pmol 70S ribosomes. Before
hybridization with the antisense(DNA) 10 pmol 70S were incubated with 15 pmol
AcPhe-tRNA and 100 [mu]g poly(U) in 25 [mu]l for 30 min at 30oC. 350 pmol of the indicated oligo(DNA) was added; the final
volume was 50 [mu]l. Hybridization was performed as described above. The poly(Phe) synthesis
was started by adding 30 [mu]l of a pre-incubation mix containing 150 pmol tRNA
Phe
, 15 nmol [
3
H]Phe (20 d.p.m./pmol), optimized amounts of S100 enzymes, 0.2 mM GTP, 2 mM ATP, 4 mM PEP and 3 [mu]g pyruvate kinase.
The poly(Phe) synthesis was linear for at least 60 s with an average rate of one
to two incorporated phenylalanine molecules per ribosome and second.
matSequencing
RNA sequencing followed the protocols of Hartz
et al
. (
24
).
RESULTS
The helix 95 and its loop from 23S rRNA are known as the [alpha]-sarcin stem-loop structure or [alpha]-sarcin domain, since the RNase [alpha]-sarcin cleaves specifically the
phosphodiester bond after G2661 (
E.coli
numbering) in that loop (
10
,
11
). The [alpha]-sarcin domain extends from C2646 to G2674
and contains the universally conserved dodecamer in the loop which is shown together with the antisense DNAs
used in this study in Table
1
. The following nomenclature to identify the probes was used: The oligo no. 1
(2660-16), for example, is a 16 nt oligo(DNA) and A2660 is the first (5'-) nucleotide of the [alpha]-sarcin domain to which the oligo can hybridize.
Saturation curves of three representative oligo(DNA)s are shown in Figure
1
, namely oligo no. 1 (complementary to the 3' half of the stem + loop of the [alpha]-sarcin stem-loop structure), oligo no. 7 (complementary to
the loop) and oligo no. 4 (complementary to the loop + 5' half of the stem). The hybridization reached a plateau between 0.3 and
0.4 oligomer per 70S ribosome at a molar excess 30 or higher of the oligo(DNA)
over ribosomes. The probes complementary to the loop or the 3'-end of the stem including the loop reproducibly hybridized a little
better (~0.4 molecules per ribosome) than those complementary to the 5'-end of the stem including the loop (~0.3).
DISCUSSION
The [alpha]-sarcin domain with its universally conserved dodecamer in the loop
is an important functional center of the ribosome. As mentioned in the
Introduction one cleavage of a phosphodiester bond of the 23S rRNA after G2661
(
E.coli
numbering) in the loop abolishes the activity of ribosomes from all kingdoms (
26
). The result is a specific block of all reactions depending on both elongation
factors, whereas all other ribosomal functions, for example the EF-G independent translocation, are not affected at all (
11
). EF-G blocked on the ribosome by fusidic acid prevents cleavage of the RNase [alpha]-sarcin (
12
), and both elongation factors protect the loop against chemical modifications (
13
). Accordingly, it is thought that this region is part of the binding and/or recognition site of
both elongation factors. Therefore, one might expect dramatic effects on
protein synthesis when an antisense DNA is hybridized to the conserved loop
region. Indeed, a severe block of the protein synthesis upon a microinjection
of an antisense oligo(DNA) against the loop was observed
in vivo
(
16
)
.
However, in this study we cannot reproduce dramatic effects caused by
hybridization against various regions of the [alpha]-sarcin domain. This failure is astonishing, since significant
hybridization of up to 0.4 antisense(DNA) per 70S ribosome was achieved. The
hybridization occurred almost exclusively at the [alpha]-sarcin domain as demonstrated with naked 23S rRNA (Fig.
2
B) and by sequencing the resulting fragment (Fig.
3
). Hybridization to isolated 23S rRNA is a stronger criteria for specificity as
compared with that to 50S subunit, since (at least in isolated 23S rRNA)
sequences outside the [alpha]-sarcin domain are also accessible which are complementary to 6 or
more nucleotides at a stretch of the antisense DNAs. About six such
complementary sequences were found per antisense DNA outside the [alpha]-sarcin domain (not shown). The specificity of hybridization to 70S
ribosomes was confirmed by the observation that all complementary probes
effectively inhibited the specific cleavage of the RNase [alpha]-sarcin (Fig.
4
B). The only exception of the set of complement probes was oligonucleotide no. 5
(2647-15), which covers the [alpha]-sarcin domain just including G2661 but not passing the
cleavage site of [alpha]-sarcin which cleaves after G2661. This oligonucleotide hybridizes
with the same efficiency as the other complementary probes. The failure of this
probe to inhibit the activity of the RNase [alpha]-sarcin provides evidence that [alpha]-sarcin approaches its cleavage site from the 3'-half of the loop of the [alpha]-sarcin domain.
The activity of our ribosome preparation is 70-100% as shown by tRNA binding assays in the presence of heteropolymeric
mRNA (
27
), whereas the hybridization of up to 0.4 molecule per 70S ribosome completely
blocks the specific cleavage of [alpha]-sarcin. It follows that [alpha]-sarcin can cleave the rRNA of only a fraction of
ribosomes probably in a distinct functional state, and that precisely this
fraction is also able to hybridize with the tested antisense DNAs. In a report
where the structure of the [alpha]-sarcin domain in solution was solved with NMR the authors mentioned
evidence that in ribosomes the reported structure co-exists with alternative structures (
28
,
29
). The solution structure described impresses as an apparently compact structure
stabilized by various non-canonical base-base interactions and containing an unusual bulged
nucleotide which is important for the action of the RNase [alpha]-sarcin (
29
). Nevertheless, the almost quantitative hybridization of the various oligos to
isolated 23S rRNA indicated by the effective RNase H digestion (Fig.
2
B) demonstrates that the structure can be easily disturbed, thus allowing
hybridization with high yields. In 70S ribosomes the saturation of
hybridization shows that the [alpha]-sarcin domain adopts a weak structure accessible for hybridization
in at least a ribosomal subpopulation of up to 40% (Fig.
1
).
.
Poly(Phe) synthesis in the presence of various antisense DNAs
Temp. (oC)
Phe incorporation
No oligo
Oligo 2
Oligo 4
Oligo 7
Oligo 8
Phe/70s
% inhib.
Phe/70s
% inhib.
Phe/70s
% inhib.
Phe/70s
% inhib.
Phe/70s
% inhib.
37
27.4 +- 1.4
0
26.8 +- 0.9
2.3
27.8 +- 1.1
0
27.2 +- 0.9
0
27.8 +- 0.7
0
30
15.2 +- 0.7
0
13.0 +- 0.7
15
13.3 +- 0.5
12
12.9 +- 0.5
15
16.1 +- 0.3
0
25
9.6 +- 0.7
0
6.2 +- 0.6
36
6.6 +- 0.6
31
6.3 +- 0.3
35
9.9 +- 0.5
0
% inhib., inhibition in percent relative to the sample without oligo(DNA) at the
same temperature. The incubation time was 45 s. Average values and deviations
from the average are given.
The observation that hybridization of the complementary probes to 70S ribosomes
effectively blocks [alpha]-sarcin cleavage but provokes only faint bands in the presence of
RNase H means that the [alpha]-sarcin domain is-at least in a distinct ribosomal state-well exposed for hybridization and attack of the
RNase [alpha]-sarcin, but that the access of RNase H is severely restricted. The
efficiency of blocking the [alpha]-sarcin activity was the same regardless whether the probe (15 nt)
covered the whole loop [oligo 7 (2653-15)] or only 8 nt of the 3'-half of the loop and 8 nt of the adjacent 3'-strand of the stem [oligo no. 1 (2660-16), Fig.
4
B]. Oligo(DNA)s complementary to the 5'-strand of the stem and the 5'-half of the loop hybridized only a little less
efficiently. It is obviously feasible to hybridize against sequences of the
stem of the [alpha]-sarcin domain which has to be melted for this hybridization. The
hybridization of oligo no. 2 to the 3' strand of the stem of the [alpha]-sarcin domain could be explicitly confirmed with naked 23S
rRNA (Fig.
3
). Our preparation of 23S rRNA maintains important structural features of the
23S rRNA within the ribosome: (i) the 23S rRNA preparation is fully active in
total reconstitution assays, whereas 23S rRNA unfolded in the presence of 6 M
guanidinium hydrochloride is inactive (F. Dohme and K. H. Nierhaus,
unpublished); (ii) unstructured RNA is totally and unspecifically degraded by
the RNase [alpha]-sarcin (
9
), whereas our 23S rRNA preparation shows only two bands, the major one is
related to the authentic [alpha]-sarcin fragment (
11
) which was also observed with 23S rRNA from
E.coli
by others (
30
). Taken together these findings argue for the possibility that the stem of the [alpha]-sarcin domain might be (at least transiently) melted in the course
of an elongation cycle
in vivo
.
Our data suggest that the structure of the [alpha]-sarcin domain might be a highly dynamic one in the course of
protein synthesis and probably of a single elongation cycle, and that the
structural changes might include melting of the stem. This conclusion explains
the universally conserved low stability of the stem of the [alpha]-sarcin stem as compared to a stem structure of the 23S-type rRNA of comparable size (
31
). The feature of dynamic structural transitions of the loop of the [alpha]-sarcin domain has been noted already when the structural and
sequence requirements for the actions of [alpha]-sarcin and ricin were compared. Ricin is an
N
-glycosidase which inactivates ribosomes by depurinating A2660 adjacent to
the G2661. The structural requirements of both enzymes were strikingly
different suggesting that the loop of the [alpha]-sarcin domain of the 23S-type rRNA can adopt different structures which might be
related to different functional states (
32
).
The dynamic changes of the [alpha]-sarcin domain during protein synthesis might explain the
disappointing effects of hybridization on the ribosomal functions. The action
of [alpha]-sarcin is hardly blocked at 37oC by the complementary probes but severely impaired at 30oC whereas little effects are seen during poly(Phe)
synthesis at the latter temperature. This differential temperature effects of
the activity of the RNase [alpha]-sarcin and the hybridization efficiency contrasts with the evidence
that the same (functional?) fraction of ribosomes is target for [alpha]-sarcin attack and hybridization (see above). A possible explanation
of these puzzling observations is that not necessarily a transition to a
distinct and different structure of the [alpha]-sarcin domain occurs at the higher temperature (37oC) but rather an acceleration of the rates of switching
between different conformations which increase the dissociation rates of the
oligonucleotides, thus facilitating the fast attack of [alpha]-sarcin (complete cleavage after 30 s, Fig.
4
A) and impairing the slow process of hybridization during the relatively fast
protein synthesis. This interpretation is also supported by the temperature
dependence of the inhibition of the poly(Phe) synthesis. No inhibition is seen
at 37oC, slight inhibition of 13 +- 3% at 30oC and modest but significant inhibition of 33 +- 3% at 25oC (Table
2
). At 30oC the inhibition is nearly complete concerning the action of [alpha]-sarcin in contrast to the slight effects observed in the
poly(Phe) synthesis. The latter probably promotes the structural transitions of
the [alpha]-sarcin domain and therefore actively expels the hybridized
antisense DNA which therefore blocks much less efficiently protein synthesis
than the action of [alpha]-sarcin.
In conclusion, we demonstrate that: (i) antisense DNA can hybridize with
practically the complete 3" strand of the stem of the [alpha]-sarcin domain; (ii) antisense DNA hybridizes to up to 40% of
the ribosome population and that the identical fraction is susceptible to the
attack of the RNase [alpha]-sarcin; (iii) the RNase [alpha]-sarcin approaches its cleavage site from the 3' half of the loop of the [alpha]-sarcin domain; (iv) the action of
the RNase [alpha]-sarcin can be blocked by antisense DNA at 30oC but not at 37oC; and (v) the poly(Phe) synthesis is also strongly
impaired at lower temperatures by antisense DNA. These results strongly support
the view noted by
Glück
et al
. (
32
, for review see ref.
10
) that the loop of the [alpha]-sarcin domain can adopt different structures. Our data indicate
further that even melting the stem of the [alpha]-sarcin domain might be involved in these structural changes, which
probably influence, control or even regulate the functional transitions of the
ribosome during protein synthesis.
ACKNOWLEDGMENTS
We thank Dr R. Brimacombe and Trixi Röhrdanz for help and discussion. The work was supported by the European Community, contract no.
CIPA CT 93-0263.
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