From the Department of Biochemistry, North Carolina State University, Raleigh, North Carolina 27695-7622
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ABSTRACT |
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Structural analysis of the 16 S rRNA in the 30 S
subunit and 70 S ribosome in the presence of
ribosome-specific antibiotics was performed to determine whether
they produced rRNA structural changes that might provide further
insight to their action. An UV cross-linking procedure that determines
the pattern and frequency of intramolecular 16 S RNA cross-links was
used to detect differences reflecting structural changes. Tetracycline
and spectinomycin have specific effects detected by this assay. The
presence of tetracycline inhibits the cross-link C967×C1400
completely, increases the frequency of cross-link C1402×1501 twofold,
and decreases the cross-link G894×U244 by one-half without affecting
other cross-links. Spectinomycin reduces the frequency of the
cross-link C934×U1345 by 60% without affecting cross-linking at other
sites. The structural changes occur at concentrations at which the
antibiotics exert their inhibitory effects. For spectinomycin, the
apparent binding site and the affected cross-linking site are distant
in the secondary structure but are close in tertiary structure in
several recent models, indicating a localized effect. For tetracycline,
the apparent binding sites are significantly separated in both the
secondary and the three-dimensional structures, suggesting a more
regional effect.
A variety of antibiotics interact with the ribosome to inhibit
protein synthesis within bacterial and eukaryotic cells (1). The point
of interruption in the translation cycle has been determined for some
antibiotics (2), making them useful in vitro to investigate the nature of elongation in protein synthesis. Some of these
antibiotics have been footprinted on the ribosomal RNAs in 30 S and 50 S subunits and in the 70 S ribosome (3) as well as on model oligomers designed to mimic local regions of 16 S rRNA (4, 5). Sites of action
have also been established by affinity and photoaffinity experiments
(6, 7). The binding sites frequently correspond to regions of the
ribosome that are implicated by other experiments in ribosome function.
In many cases, binding site assignments are supported by
resistance-conferring mutations in 16 S and 23 S rRNAs (2, 8).
Antibiotics, either directly by binding or indirectly through
conformation alterations, must result in the inability of tRNA or
translation factors to bind the ribosome or else inhibit some processes
needed during translation. However, little is known regarding whether
conformational perturbations accompany binding, even in instances in
which detailed information supporting binding sites and the nature of
translation interruption are known.
UV cross-linking of the rRNA in the ribosome provides an opportunity to
monitor changes in rRNA conformation and, consequently, the ribosome
global structure. We have previously determined the identity of 14 UV-induced cross-links in 16 S rRNA within the 30 S subunit (9) and 15 cross-links in the 70 S ribosome (10). Because of the gel
electrophoresis method used in the detection, all of these cross-links
occur between nucleotides that are distant in the primary sequence.
These cross-links occur because the partner nucleotides possess a
suitable distance and geometry during the lifetime of the excited state
(on the order of 1 µs; Ref. 11); therefore, their frequency provides
a method to screen substrates and other agents for their ability to
affect ribosomal conformation.
In this report, UV irradiation was repeated in the presence of 13 antibiotics to determine whether they produce measurable changes in the
ribosome structure that might be related to their activity. These 13 antibiotics were known to bind either the 30 S or 50 S subunit. Some of
these antibiotics have localized binding sites on 16 S rRNA, as
determined by chemical probing, that are adjacent in the 16 S rRNA
secondary structure to nucleotides that participate in cross-links and
may affect the frequency of such contacts. Two of these antibiotics,
spectinomycin and tetracycline, have discernible effects on the
frequency of specific UV cross-linking sites in 16 S rRNA. The
implications of these effects with respect to the antibiotic action are discussed.
Preparation of Ribosomes and UV Cross-linking
Procedures--
Escherichia coli 70 S ribosomes and
ribosomal subunits were prepared according to Makhno et al.
(12) and dissolved in CMN1
buffer. In some experiments, CMN buffer was used with Mg2+
concentrations from 0.5 to 50 mM. 70 S ribosomes were
prepared by reassociating equimolar amounts of 30 S and 50 S subunits
and were free of mRNA or tRNA. Samples were incubated with the
following concentrations of antibiotics (Sigma) that were previously
determined to produce specific footprints in the 16 S rRNA or 23 S rRNA
by chemical probing (2, 3): (a) 5 × 10
Cross-linked 16 S rRNA was separated by gel electrophoresis on gels
made with 3.6% acrylamide:bisacrylamide (70:1), 8.3 M urea, and BTBE buffer as described previously (9). For analysis of
cross-linking sites, the location of the bands containing
un-cross-linked and cross-linked 16 S rRNA were detected with a
PhosphorImager, and bands were cut out and eluted by
ultracentrifugation through cushions containing 2 M CsCl
and 0.2 M EDTA, pH 7.4, for 12 h at 40,000 rpm (13).
RNA pellets were redissolved in 250 µl of H20,
phenol-extracted, and re-precipitated before further analysis.
Determination of the Identity and Frequency of Cross-linked
Sites--
The cross-linking sites in separated 16 S rRNA were found
by primer extension analysis using 11 DNA primers complementary to
regions throughout 16 S rRNA (9). The frequency of cross-linking was
determined from PhosphorImager data (ImageQuant; Molecular Dynamics
Inc.) of duplicate independent experiments. To normalize for RNA
loading, cross-link band intensity was referenced to the same
cross-link band (C54×A353) in each respective lane. This reference
band showed <10% variance in all lanes when referenced to the
un-cross-linked 16 S rRNA parent band in the same lane.
Six classes of antibiotics were examined in these experiments:
(a) aminoglycoside (neomycin, paromomycin, streptomycin,
gentamycin, kanamycin, spectinomycin, and hygromycin), (b)
tetracycline, (c) macrolide (erythromycin), (d)
peptide (thiostrepton and viomycin), (e) fusidic acid, and
(f) chloramphenicol. The seven aminoglycosides, tetracycline, and the two peptide antibiotics are thought to bind primarily to the 30 S subunit, and the two peptide antibiotics also
bind to the 50 S subunit (2). Erythromycin and chloramphenicol bind
exclusively to the 50 S subunit, and fusidic acid prevents the release
of elongation factor G·GDP from the ribosome (2). These last three
antibiotics were included to test the possibility that an alteration in
the 50 S subunit structure may induce changes in the structure of the
30 S subunit, within the 70 S ribosome. Both 30 S and 70 S ribosomes
were irradiated in the presence of the listed antibiotics, and, with
exceptions noted below, the effects were identical. In all instances in
these experiments, empty ribosomes were used to avoid heterogeneity in
the ribosomal state and to avoid the complication of possible
substrate-mediated structure changes. In addition, previous
characterizations of antibiotic binding sites (3, 7) and our
identification of UV cross-links in 16 S rRNA (9) have been performed
on empty ribosomes.
Tetracycline and spectinomycin were the only compounds tested
that affected the cross-linking pattern of 16 S rRNA within 30 S
subunits or 70 S ribosomes (Fig. 1). At
2.5 × 10
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
6
M neomycin, (b) 5 × 10
6
M paromomycin, (c) 5 × 10
6
M streptomycin, (d) 1 × 10
4
M gentamycin, (e) 1 × 10
4
M kanamycin, (f) 1 × 10
4
M spectinomycin, (g) 2.5 × 10
4 M tetracycline, (h) 5 × 10
6 M erythromycin, (i) 5 × 10
6 M thiostrepton, (j) 5 × 10
6 M fusidic acid, (k) 5 × 10
6 M chloramphenicol, (l) 5 × 10
6 M viomycin, and (m) 1 × 10
4 M hygromycin. Concentration series
have also been investigated for tetracycline and spectinomycin. Samples
were incubated for 30 min at 37 °C, placed on ice for 10 min, and
irradiated at 4 °C for 20 min in a quartz cuvette with continuous
stirring. Irradiation was performed with a 312 nm trans-illuminator
(Fotodyne Corp.) as described previously (9). Sample concentrations
were usually 1 µg RNA/µl, with the exception of samples for
preparative separation, which were irradiated at 6 µg RNA/µl. RNA
was recovered from the samples by proteinase K digestion, phenol
extraction, and ethanol precipitation. The RNA was dephosphorylated
with shrimp intestinal phosphatase and purified by proteinase K
digestion, phenol extraction, and ethanol precipitation. 16 S rRNA was
then isolated on a 1% agarose gel before 5' end-labeling with
[
-32P]ATP by T4 polynucleotide kinase.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
4 M, the concentration used for
chemical footprinting experiments (3), tetracycline affected three
identified cross-links and one incompletely identified cross-link. The
third band from the top of the pattern, which was verified as
C967×C1400, was completely inhibited. In this and other gels,
there was some decrease in the intensity of the topmost band in
the pattern, which was shown to contain U244×G894. The third band from
the bottom of the gel, which was shown to contain the cross-link
C1402×C1501, increased twofold in intensity in both 30 S subunits and
70 S ribosomes. The fourth band from the top also decreased in the
presence of tetracycline. U534 is one part of this cross-link, but we
have been unable to find a partner for it. At 1 × 10
4 M, the concentration used for chemical
footprinting experiments (3), spectinomycin decreased the intensity of
the cross-link found in the second band from the top (Fig. 1), which
was shown to contain the cross-link C934×U1345, by 60%.
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Fig. 1.
Comparison of cross-links induced by UV
irradiation in 16 S rRNA in 30 S subunits and 70 S ribosomes in the
presence of spectinomycin and tetracycline. Lanes 30S
and 70S indicate experiments performed with 30 S subunits or
70 S ribosomes, respectively. Control indicates experiments
performed in the absence of antibiotics. Tet or
Spec indicates experiments performed in the presence of
tetracycline or spectinomycin, respectively. Concentrations used were
1 × 10 4 M spectinomycin and 2.5 × 10
4 M tetracycline. Affected cross-links are
indicated by arrows. The asterisk indicates the
band containing the cross-link involving U534. Labels to the
right of the figure indicate fractions taken in the
preparative experiments used in Fig. 2 and 3.
Reverse transcription analyses of the regions of 16 S rRNA containing
the cross-links affected by tetracycline are shown in Fig.
2. A and B show the
results of reverse transcription in the nucleotide intervals 212-265
and 866-914, respectively, in which differences were seen in the
tetracycline sample. In lane 7 in each panel, the stops
indicating cross-links at U244 and G894 decreased in the tetracycline
sample relative to the control sample. C and D
show the results of reverse transcription reactions in the nucleotide
intervals 952-983 and 1387-1409. Lane 5 of each panel
contains stops indicating that the cross-link C967×C1400 disappeared.
E and F show the results of reverse transcription reactions in the nucleotide intervals 1390-1411 and 1491-1504. Lane 2 of each panel contains stops indicating that the
cross-link C1402×C1501 increased in the presence of tetracycline
relative to the control. The effects of tetracycline on cross-link
intensity are the same in both 30 S subunits and 70 S ribosomes.
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Reverse transcription analysis of the cross-linked band affected by
spectinomycin is shown in Fig. 3. The
only reverse transcription stops affected by spectinomycin were in
intervals 907-953 (A) and 1330-1365 (B). A
decrease in the stops at 935 and 1346 in lane 6 of each
panel indicates that the cross-link C934×1345 is affected. The
decreases in intensity are consistent with the difference seen in
the analytical gel pattern (Fig. 1).
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Titration experiments were performed with tetracycline to determine the
concentration threshold on the 16 S rRNA tertiary structure (Fig.
4). At concentrations between 2.5 × 104 M and 1 × 10
5
M, tetracycline specifically affects the cross-links noted
above. There was no change in the frequency of any of the cross-links before the low concentration limit was reached. In the sample irradiated in the highest concentration of tetracycline, several cross-links were partially or completely inhibited; this is attributed to the nonspecific binding of tetracycline to the ribosome and/or a
decrease in cross-linking due to the high tetracycline absorbance.
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Titration experiments were also performed at five spectinomycin
concentrations (Fig. 4). These show that the decrease in the C934×U1345 cross-link is seen at or above 1 × 106
M spectinomycin, with a complete recovery of cross-link
frequency below 1 × 10
6 M.
The effects of tetracycline and spectinomycin were determined in 30 S subunits and 70 S ribosomes at Mg2+ concentrations from 0.5 to 50 mM. Mg2+ has been shown to govern the frequency of cross-linking of several cross-links including C967×C1400 and C1402×C1501 (10). C967×C1400 was inhibited at all Mg2+ concentrations, except that in 0.5 mM Mg2+ in 30 S ribosomes, the frequency of the cross-link, even in the control sample, was too low to detect. The increase in the frequency of C1402×C1501 due to tetracycline was seen in 30 S subunits and 70 S ribosomes at all Mg2+ concentrations above 5 mM. Below 5 mM, no increase in C1402×C1501 could be seen (data not shown). The decrease in frequency of C934×U1345 due to spectinomycin did not change over the stated Mg2+ concentration range (data not shown).
Several cross-links, C1400×C1501, C1402×C1501, and C1397×C1497,
identified in the decoding region of 16 S rRNA nearly co-migrate in gel
electrophoresis (10). There was a possibility that neomycin and
streptomycin would affect the distribution of conformations in that
region. However, no detectable changes in the analytical gel
electrophoresis experiments involving these antibiotics were seen, nor
were any changes seen in primer extension experiments (data not shown).
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DISCUSSION |
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Of the 13 antibiotics examined in this study, tetracycline and spectinomycin showed specific and different structural effects in 16 S rRNA that were detectable by the UV cross-linking assay. The inhibition of cross-links by these antibiotics correlates well with their known effective concentrations (14, 15), indicating that the 16 S rRNA tertiary structure effects are linked to the loss of small subunit function during translation. For the tetracycline response, all three of the cross-links are affected at the same tetracycline concentration.
Tetracycline has been shown to inhibit protein synthesis by interfering with the binding of aminoacyl-tRNA to the ribosomal A-site (2, 14), but it does not prevent the binding of tRNAPhe to the P-site (16). However, tetracycline has been reported to interfere with initiation factor-dependent tRNAfMet binding (2, 17). In a study in which the binding of a fluorescent analogue of tetracycline, demeclocycline, was monitored, displacement of demeclocycline by tRNAfMet and by A-site tRNAPhe binding was confirmed (18). Tetracycline did have an inhibitory effect on the P-site when the determination was done at high tRNA:70 S stoichiometries, and this was attributed to a fraction of ribosomes that bind tRNA in the P-site with lower affinity (19).
Photochemical cross-linking experiments have shown that tetracycline
cross-links primarily to protein S 7 and, to a lesser extent, S 18 and
S 4 (20). S 7 has been footprinted by both base modification and
Fe2+/EDTA cleavage experiments to the lower part of domain
III in 16 S rRNA (21). More recent experiments have determined that with tetracycline concentrations lower than 4 × 105
M, higher UV light flux, and shorter irradiation times (30 s), tetracycline cross-linked to 16 S rRNA itself at nucleotides G693 (near helix 23b), G1300, and G1338 (both of them near helix 42) (7). At
concentrations of
1.2 × 10
4 M,
additional cross-linking sites, including one at G890, are seen (7). In
experiments done at 1 × 10
4 M,
tetracycline produced a strong chemical protection footprint in 16 S
rRNA at nucleotide A892 and a weak reactivity enhancement of
nucleotides U1052 and C1054 (3) (see Fig.
5). Recently, a C
G substitution at
position 1058 granting resistance to tetracycline was reported (22),
which also indicates that this region is connected to tetracycline
function.
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Footprints for tRNA in the P-site include C1400, G1338, C967, and G693 (23). Because two of the three affected UV cross-links and all three tetracycline-RNA cross-links are associated with the tRNA P binding site, it is worthwhile to consider how tetracycline might interfere with A-site tRNA binding. Rodnina et. al. (24) have investigated EFTu·aminoacyl tRNA·GTP binding and found that the rate and strength of the initial complex and the codon recognition complex were both enhanced by cognate tRNA bound to the P-site. This suggests an effect by the P-site-bound tRNA or some change in the ribosome that depends on P-site occupancy that influences the ternary complex-ribosome interaction. Therefore, alteration of the P-site by tetracycline may not affect the binding of tRNA in the P-site itself but may inhibit some conformational adjustment needed for tRNA binding to the A-site.
The reported antibiotic binding sites (3, 7) and the affected RNA
cross-linking sites are compared in a 16 S rRNA three-dimensional model
(Fig.
6),2
which shows the locations of the helices containing the tetracycline and spectinomycin binding sites and the RNA-RNA cross-links affected by their binding. This model is different from recent models
(26-28) in several regions because it incorporates new information
including the UV cross-links C967×C1400 (9, 10), U793×G1517, and
G976×G13613 and
site-specific psoralen cross-links
(25).4
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Approximate distances from G1300, G1338, and G693 to C1400 (the P-site) are 45, 35, and 25 Å, respectively, and distances from G1300, G1338, and G693 to A1408 (associated with the A-site) are 57, 50, and 36 Å, respectively. In another recently proposed model (26), the distances to C1400 are 67, 40, and 81Å, and the distances to A1408 are 95, 70, and 82 Å. In both models, the distances between the sites of tetracycline-RNA cross-linking (7) and the P-site nucleotide C1400 (3) are less than those to the A-site nucleotide A1408 (23). The molecular dimensions of tetracycline measure approximately 8 × 12 Å.
Spectinomycin causes a structural change in domain III that
results in a decrease in the frequency of cross-link C934×U1345. Spectinomycin causes a strong protection from chemical reactivity at
C1063 and G1064 (see Fig. 5) and a weak enhancement of reactivity at
G973 (3). In addition, spectinomycin resistance is conferred by a
CA, G, U mutation at position 1192 in 16 S rRNA. Because overexpression of a fragment of helix 34 corresponding to nucleotides 1047-1067 and 1189-1210 (linked by a hairpin loop) also confers spectinomycin resistance (2), the binding site is most likely located
in helix 34. In addition, it was shown that susceptibility to
spectinomycin could be restored in C1192 mutants by an additional U1351
C mutation in the top of helix 43 (15). The partial inhibition of cross-link C934×U1345 by spectinomycin supports evidence for an
interplay between helix 34b and 43. The helix that contains the
spectinomycin footprint at C1063 (helix 34b) and the nucleotides that
form the C934×U1345 cross-link at the end of helix 28 are highlighted
in the model (Fig. 6). The distance between these two regions is 12 Å in the model shown (Fig. 6), compared with 20 Å in the Mueller and
Brimacombe (26) model. A surprising result is that spectinomycin has no
effect on the U1052×C1200 cross-link, which is in close proximity to
both the resistance mutation site (C1192) (3) and the proposed binding
site in 16 S rRNA. In addition, there are no changes in the cross-links U1052×C1200, A1093×G1182, or U1126×C1281, all of which are located in domain III. This indicates that spectinomycin is not producing a
global rearrangement of the domain III tertiary structure but rather
some specific alteration of the structure near the junctions of helices
28, 29, and 43. It is known that spectinomycin interferes with
elongation factor G binding during early rounds of protein synthesis
(2). It has recently been shown that elongation factor G interacts with
the 30 S subunit near the 1338 region (29); therefore, it also possible
that the structural perturbation caused by spectinomycin alters
elongation factor G association.
The neomycin-type aminoglycosides (hygromycin, gentamycin, neomycin,
and paromomycin) protect 16 S rRNA nucleotides A790, A791, A909, A1394,
A1413, and G1487 and enhance reactivity at C525 (3). These antibiotics
are generally thought to induce miscoding by inhibiting A-site
occupation and to inhibit translocation (2). Streptomycin causes
miscoding (2) and protects 16 S rRNA nucleotides 911-915, nucleotides
in the 1408-1418 and 1482-1494 regions, and nucleotide 1468 in 30 S
subunits in 20 mM Mg2+ (3, 30) and binds to
naked 16 S rRNA fragments of the decoding region in 20 mM
Mg2+. Viomycin and thiostrepton induce protections in both
16 S and 23 S rRNA (for viomycin, these are known to be protections at nucleotides 912-915), and both antibiotics also inhibit A-site occupation and cause miscoding (2). It is thought that their binding
restricts the ribosome conformation, thereby preventing A-site
occupation or translocation (2). None of those antibiotics produces an
observable effect in this experiment, so it is possible that these
antibiotics interact with only a local region of the ribosome.
Alternatively, the tertiary structure effects for the other antibiotics
may be too subtle to detect by the present cross-linking, may be
elicited only in the ribosome under working conditions, or may affect
parts of the ribosome not monitored by the cross-links that are made by
UV irradiation.
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ACKNOWLEDGEMENT |
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We thank Vickers Burdett for comments on the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM43237 and by a Graduate Assistance in Areas of National Need Fellowship from the U. S. Department of Education (to J. W. N.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 919-515-5703;
Fax: 919-515-2047.
2 M. A. Dolan, P. Babin, and P. Wollenzien, unpublished data.
3 T. Shapkina, unpublished data.
4 D. Mundus, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are:
CMN, 80
mM cacodylic acid, pH 7.5, 20 mM
MgCl2, 100 mM NH4Cl, and 4 mM -mercaptoethanol;
BTBE, 30 mM
bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane, 30 mM
boric acid, and 2.5 mM EDTA, pH 6.8.
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