From the Department of Biochemistry and Genetics, The
University of Newcastle, Newcastle upon Tyne, NE2 4HH, United Kingdom
and the § Astbury Centre for Structural Molecular Biology,
Faculty of Biological Sciences, The University of Leeds, Leeds, LS2
9JT, United Kingdom
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ABSTRACT |
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The secondary structure of an RNA aptamer, which
has a high affinity for the Escherichia coli MetJ repressor
protein, has been mapped using ribonucleases and with diethyl
pyrocarbonate. The RNA ligand is composed of a stem-loop with a highly
structured internal loop. Interference modification showed that the
bases within the internal loop, and those directly adjacent to it, are important in the binding of the RNA ligand to MetJ. Most of the terminal stem-loop could be removed with little effect on the binding.
Ethylation interference suggests that none of the phosphate groups are
absolutely essential for tight binding. The data suggest that the MetJ
binding site on the aptamer is distinct from that of the natural DNA
target, the 8-base pair Met box.
Several genes involved in the biosynthesis of methionine in
Escherichia coli are transcriptionally regulated by the
methionine repressor, MetJ (1). The basic interaction occurs between a homodimer of the 12-kDa MetJ repressor subunits and an 8-base pair
sequence that constitutes a Met box (2). The Met box is a tandem repeat
that occurs between two and five times in natural operators and to
which additional repressor dimers bind in a cooperative manner (3). The
protein has a Over the last decade in vitro selection and amplification
techniques have been developed to isolate tight binding nucleic acid
ligands for a wide range of target molecules (7, 8). RNA molecules in
particular have been isolated for a diverse set of targets, including
proteins (9), amino acids (10), and nucleotides (11). These RNA
aptamers potentially have very widespread applications as lead
compounds in therapeutic situations, e.g. RNA-based
inhibitors of the type I human immunodeficiency virus (HIV-I) reverse
transcriptase (12, 13) or as ligands in diagnostic kits and biosensors.
Despite their importance we have relatively little structural
information on the ways in which aptamers interact with protein
targets. Recently, one of our laboratories reported the first x-ray
crystal structure for an aptamer bound to the RNA bacteriophage MS2
coat protein (14). This structure revealed that the aptamer is bound to
the natural RNA binding site of the protein, and although being based
on differing primary and secondary structures, the aptamer is able to
mimic most of the key recognition contacts of the natural ligand.
To extend our knowledge of the way in which aptamers interact with
their protein targets, we have isolated tight binding RNA ligands for
the E. coli MetJ repressor, a sequence-specific DNA-binding protein with no known RNA-binding function.2 Previously, we
have shown that systematic evolution of ligands by exponential
enrichment (SELEX) with degenerate DNA ligands results in isolation of
tight binding DNA molecules based on the Met box consensus (16), a
situation analogous to the isolation of RNA aptamers for the MS2 coat
protein (9). However, a high proportion of the SELEX-RNA ligands
isolated by binding to MetJ contained the consensus sequence
5'-UGCAUACUCGUUN(3-16)A(G)GCAUUGCAGCA-3',2
which is unrelated to the Met box DNA target. Remarkably, several of
these ligands bind more tightly to an apo-MetJ dimer than does the DNA
consensus, a two Met box site interacting with two cooperatively bound
holorepressors (3). Therefore, it is of interest to determine how such
RNA aptamers interact with MetJ. Do they mimic the binding of the
natural DNA Met box (as for the MS2 coat protein with RNA ligands and
MetJ with DNA ligands), or alternatively, do they represent novel
solutions to the MetJ recognition problem? Inhibition of DNA binding
proteins by RNA aptamers is likely to be of very general interest.
DNA-binding proteins constitute a large group of potential drug
targets, and RNA SELEX offers a route to generate tight binding
inhibitors of such molecules.
Here a number of techniques have been employed both to determine the
solution structure of the highest affinity RNA ligand and to probe the
nature of the interaction between the two macromolecules. In particular
we have attempted to determine the regions of the RNA aptamer that are
involved in binding to the protein. Similar approaches have been used
to good effect to study the inhibition of HIV-I reverse transcriptase
with RNA ligands (17). In the absence of high resolution structural
data for the RNA-protein complex, these studies provide a useful
insight into how the aptamer complex differs from the DNA operator
complex with the same protein.
Oligoribonucleotide Synthesis and Purification--
RNA was
synthesized on an Applied Biosystems 391 synthesizer using
t-butyldimethylsilyl RNA-phosphoramidites (ChemGenes, Stoke on Trent, UK). Ancillary RNA synthesis reagents were supplied by
Applied Biosystems (Warrington, UK). The standard 1.0-µmol DNA
synthesis cycle was used with a longer coupling and capping periods of
360 s and 40 s, respectively. Syntheses were carried out
trityl-off. Protecting groups, other than the silyl group, were removed
by treatment with 2 ml of fresh methanolic ammonia at 37 °C for
20 h. Desilylation was performed by dissolving the RNA in 500 µl
of dimethyl sulfoxide (Aldrich) and treating the solution with 500 µl
of triethylamine trihydrofluoride (Aldrich) at room temperature for
48 h. The RNA was desalted on a NAP-25 column (Amersham Pharmacia
Biotech) prior to ion-exchange high performance liquid chromatography
using a NucleoPac PA-100 column (4 × 250 mm) (Dionex, Camberley,
UK). The column was developed, at 55 °C, with an ammonium acetate
gradient (0-1.5 M, pH 7.5) in solutions containing 10%
(v/v) acetonitrile. The required fraction was dried in a rotary
evaporator resuspended in sterile water and desalted as above. RNAs
containing a hexaethylene glycol spacer were prepared as above using
18-O-dimethoxytritylhexaethylene glycol,
1-[(2-cyanoethyl)-(N,N-diisopropyl)]phosphoramidite
(Glen Research, Sterling, VA). Oligoribonucleotides containing
4-thiouridine were synthesized as above with one additional step.
Immediately following synthesis the support-bound oligomers were
treated with 1 ml of 0.3 M
1,8-diazabicyclo[5.4.0]undec-7-ene (Aldrich) in dry acetonitrile for
3 h at room temperature to remove the cyanoethyl group that
protects the thiol (18). The 1,8-diazabicyclo[5.4.0]undec-7-ene was
then carefully decanted, and the support was extensively rinsed with
acetonitrile, prior to treatment with methanolic ammonia. The
4-thiouridine phosphoramidite was synthesized as reported previously
(19). All oligoribonucleotides were stored at Radioactive Labeling of
Oligoribonucleotides--
Oligoribonucleotides were labeled, at their
5'-ends using [ MetJ Repressor--
MetJ repressor, purified as described (22,
23), was a gift from Isobel D. Lawrenson (University of Leeds). It was
supplied as an ammonium sulfate precipitate and stored at 4 °C until
required. Precipitates were dissolved in 100 mM sodium
phosphate (pH 7) and dialyzed against this buffer, prior to use. The
protein concentration was determined spectrophotometrically (22,
23).
Enzymatic and Chemical Probing of RNA Secondary
Structure--
RNA labeled at either the 5'- or the 3'-end (
Diethyl pyrocarbonate (DEPC) modification of adenosine and guanosine
was performed essentially as described in Ref. 24. Approximately 6 pg
( Interference Analysis of MetJ Repressor-RNA Complexes--
RNA
was chemically modified with either DEPC, hydrazine, or
ethylnitrosourea. Adenosine and guanosine were modified with DEPC, as
described above, using the denaturing buffer. Uridine bases were
modified by dissolving an ethanol precipitate of labeled RNA (
The modified RNA (2-4 pg) was heated at 90 °C for 2 min, then
placed at 37 °C for 15 min prior to its addition to 50 mM Tris/HCl, pH 7.0, 50 mM KCl, 10 units of
RNAguard (Amersham Pharmacia Biotech), in the presence or absence of
MetJ repressor (20 µM, dimer concentration) (final volume
20 µl). RNA binding does not require AdoMet (in contrast to the
binding of Met box DNA) and so this was omitted from both the
incubation buffer and the gel. The samples were incubated at 37 °C
for 10 min, after which 5 µl of 50 mM Tris/HCl, pH 7.0, 50 mM KCl, 50% (w/v) glycerol was added. Gel retardation of the RNA was achieved by electrophoresis on a 1.5-mm-thick 10% (w/v)
polyacrylamide gel, buffered with 10 mM sodium phosphate, pH 7.0. The samples were briefly run into the gel at 300 V, and the
gels then were run at 100 V using 100 mM sodium phosphate (pH 7.0) running buffer. Following electrophoresis the appropriate bands were excised, and the RNA was recovered by elution as described above. 25 units of tRNA (type X-SA, Sigma) was added before the RNA was
precipitated. Both DEPC- and hydrazine-modified RNAs were then cleaved
at the modified nucleotides, using aniline, as described above.
Ethylated RNA was cleaved with 0.1 M Tris/HCl, pH 9.0 (25). The cleaved products were visualized by denaturing gel electrophoresis as described above.
Filter Binding Assays--
Filter binding assays were performed
as described previously (6). The MetJ protein was serially diluted in
50 mM Tris/HCl, pH 7, 50 mM KCl and incubated
with approximately 20 nM RNA for 10 min at 37 °C.
Samples were filtered through 0.45-µm nitrocellulose filters
(Whatman) and rinsed with 2 × 400 µl of binding buffer. Following drying, 5 ml of Ecoscint A (National Diagnostics) was added,
and the samples were counted in a liquid scintillation counter. The
Kd was estimated as the protein concentration at
which half-maximal binding of the RNA occurred.
Circular Dichroism of RNA and MetJ Repressor-RNA
Complexes--
Circular dichroism spectra were obtained using a Jobin
Yvon CD6 Dichrograph with the cell (path length 10 mm) thermostatted at
37 °C, unless stated otherwise. Samples (0.15 ml) were prepared in
50 mM Tris/HCl, pH 7.0, 50 mM KCl. An RNA
concentration of 1 µM was used, with the protein
concentrations quoted in the legend of Fig. 9.
UV Cross-linking of MetJ Repressor with RNA Containing
4-Thiouridine--
Cross-linking of the MetJ repressor to RNA
containing 4-thiouridine was carried out as described previously (26).
Samples containing 7.5 µM MetJ repressor dimer and
approximately 50 nM radiolabeled oligoribonucleotide in 50 mM Tris/HCl, pH 7, 50 mM KCl, were incubated at
37 °C for 10 min prior to irradiation, at 350 nm, for 10 min. The
oligoribonucleotide shown in Fig. 1A was used, but with the
uridines at positions 20, 23, 35, and 36 replaced with 4-thiouridine.
Potential protein-RNA cross-links were analyzed by SDS-polyacrylamide
gel electrophoresis.
Secondary Structure of the RNA Ligand--
The sequence and the
predicted secondary structure of the SELEX RNA ligand used in this
study is shown in Fig. 1. It consists of
a stem-loop (terminal loop) and an internal loop (27). To test this
prediction experimentally, we have mapped the RNA ligand using
ribonucleases and with DEPC. As shown in Fig.
2, the terminal loop was most sensitive
to cleavage by the single-strand specific nucleases RNase A
((C/U)-specific) and RNase U2 (A-specific). A number of
nucleotides within and directly adjacent to the internal loop were also
cleaved by the nucleases, but to a lesser degree than those in the
terminal loop. These include
A6/U7/A8 (5'-labeled strand) and
C33/C38/C41 (3'-labeled strand). A
few internal loop nucleotides, which would be expected to be cleaved,
if single-stranded, were either not affected or were very poorly cut by
the nucleases examined. Nucleotides in this category were
C9/U10/C11 (5'-labeled strand) and
A34/U35/U36/A39
(3'-labeled strand). The nucleotide C29, which is predicted
to be in a 2-nucleotide bulge in the main stem, was also susceptible to
cleavage by RNase A. Consistent with most of the guanosines being
double-stranded, RNase T1 (G-specific) did not cleave well,
including position G37 within the internal loop. Comparison
of DEPC modification (which modifies the N7 position of
adenosine and, to a lesser extent, that of guanosine) of the RNA at
37 °C and 90 °C showed marked differences, with greater
modification under denaturing as compared with native conditions, for
two of the three adenosines (A31/A42),
predicted to be in double-stranded regions (Fig. 2). The four adenosines
(A6/A8/A34/A39) which
are predicted to be in the internal loop, were also modified to a
greater degree in the denatured oligonucleotide compared with the
native one. In contrast, little difference in reactivity was observed
for the two adenosines (A21/A22) in the
terminal loop. The nucleotide A24 also behaved in this
manner. Although predicted to be in a double-stranded region, this
nucleotide is immediately adjacent to the terminal loop.
The results are summarized in Fig. 3 and
are in broad agreement with the predicted structure. The terminal loop,
which closes the stem-loop, has properties consistent with its being
single-stranded. The regions predicted to be double-stranded map as a
duplex when probed with nucleases or DEPC. The most interesting region
is the internal loop. A number of nucleotides within this region, that
would be expected to be ribonuclease-sensitive, were either not cleaved
or were cut significantly more slowly than those in the terminal loop.
Similarly, several nucleotides have a diminished reactivity to DEPC in
the native compared with the denatured RNA. This suggests that the
internal loop cannot be considered as single-stranded but must contain
some degree of stable secondary structure, limiting the accessibility
of both ribonucleases and DEPC. It is conceivable that some of the
bases on opposite sides of the internal loop form non-canonical base
pairs. In addition bases on the same strand may be strongly
stacked.
Mapping Contacts between Bases in the RNA Ligand and the MetJ
Repressor--
Interactions between the bases in the RNA ligand and
the MetJ repressor have been determined by binding-interference
analysis. DEPC has been used to probe the role of adenosine and
guanosine, and hydrazine has been utilized for uridine. The results for
DEPC are shown in Fig. 4. To map the
entire RNA sequence, it was necessary to use both 3'- and 5'-end
labeling. A similar quality autoradiogram (not shown) was obtained
using hydrazine. Scans of both the DEPC and hydrazine autoradiograms
are shown in Fig. 5, and the results are
summarized in Fig. 6. Both DEPC and
hydrazine caused notable interference at a number of bases.
Modification of bases within, and directly adjacent to, the internal
loop significantly reduced the binding of the RNA ligand to MetJ.
Modification of the eight bases in and around the terminal loop had no
effect on binding. Modification of the bases in the intervening stem
that connects the internal and the terminal loops had a mildly
detrimental effect on binding. This strongly suggests that it is the
internal loop region of the RNA ligand that is responsible for tight
binding to the MetJ repressor.
The importance of the internal loop was confirmed using two truncated
RNA ligands. The deletions remove (a) the terminal loop and
2 base pairs from the stem and (b) the terminal loop, 5 base pairs from the stem, and the two single stranded cytosines (Fig. 1). In
both cases the deleted bases were replaced by a hexaethylene glycol
chain to substitute as a hairpin loop (28). The binding affinity of the
two shortened RNA molecules to the MetJ repressor were determined using
a filter binding assay (Fig. 7). Neither deletion markedly affected the binding of the RNA to MetJ.
Kd values (assessed as the protein-dimer
concentration at which one-half of the counts were retained on the
filter) of between 2 and 4 ± 0.5 × 10 Mapping Contacts between Phosphates in the RNA Ligand and the MetJ
Repressor--
Ethylnitrosourea, which ethylates the non-esterified
oxygen atoms of backbone phosphates in nucleic acids, was used to probe the role of the RNA phosphate groups in binding to the MetJ repressor. No single phosphate was found to be critical for interaction with the
MetJ repressor, i.e. there was no phosphate group, which
when ethylated, strongly inhibited the binding of MetJ. This is shown in Fig. 8, which shows scans of the
interference autoradiograms.
The role of RNA phosphates in binding to MetJ has been further
evaluated by the determination of Kd values at
various salt concentrations using a filter binding assay (data not
shown). If interactions between negatively charged phosphates and
positively charged amino acid side chains are important, the
Kd would be expected to increase with increasing
ionic strength (29). In 10 mM Tris/HCl, pH 7.0, with KCl
concentrations of 10, 25, 50, and 100 mM, identical
Kd values of 1.2 ± 0.3 × 10 Conformation of the RNA Ligand, Both Free in Solution and Bound to
MetJ--
Circular dichroism data (Fig.
9) are consistent with the RNA being
largely double-stranded and in an A-form conformation. The spectrum of
the free RNA shows a large positive Photocross-linking of 4-Thiouridine-containing RNA to MetJ--
It
has previously been shown that both oligodeoxyribonucleotides
containing 4-thiothymidine and oligoribonucleotides containing 4-thiouridine can be used to photocross-link DNA- and RNA-binding proteins (26, 34). We had hoped to use this approach to map the regions
of MetJ in contact with the RNA ligand. Unfortunately, substitution of
several uridines by 4-thiouridine, in the oligoribonucleotide shown in
Fig. 1A, and subsequent 350 nm irradiation of these
oligoribonucleotides, in the presence of MetJ, failed to generate any
cross-links (data not shown). Considering the data above, this is
hardly surprising for the uridines (positions 20 and 23) in the
terminal loop. The failure of 4-thiouridines, within the internal loop
(positions 35 and 36), to cross-link was disappointing, particularly as
modification of uridines 35 and 36, with hydrazine, interferes with
MetJ binding (Figs. 5 and 6). However, we have previously shown that
the proximity of 4-thiouridine to a protein is not necessarily
sufficient for cross-linking to occur (26). Filter binding (not shown)
demonstrated that replacement of uridines 20, 23, 35, and 36 with
4-thiouridine either did not affect binding or reduced it to a small
extent, such that significant amounts of MetJ-RNA complex was formed at the concentrations used for photocross-linking.
Although there is a great deal of interest in RNA ligands selected
to bind particular target molecules with high affinity, there is, as
yet, little structural information on the nature of the RNA-target
complexes. In the last few years several structures have been
determined for selected RNAs bound to small molecular weight ligands
such as ATP, using NMR spectroscopy (35). More recently the first high
resolution crystal structure for a selected RNA-protein complex has
been determined (14). Additionally, low resolution methods, mainly
interference and protection modification combined with base
substitutions, have been used to map the interfaces between proteins
and selected, high affinity, RNA ligands (17).
Here we have characterized an RNA ligand of a natural DNA-binding
protein, the E. coli methionine repressor, MetJ. The wealth of structural data on the wild-type MetJ system, including crystal structures for the apo-, holo-, and repressor-operator complexes (4,
5), suggest that this is a good model in which to study the details of
aptamer-protein recognition and to compare how the same protein can
interact with both RNA and DNA. So far MetJ has proven to be relatively
straightforward to crystallize, and hopefully it will be possible to
obtain an x-ray structure for this protein in complex with an RNA
aptamer. Additionally the small size of MetJ, a homodimer of subunit
molecular mass 12 kDa, suggests that NMR spectroscopy might be useful,
and the Leeds group already has extensive NMR assignments of the
repressor protein. The secondary structure of the free RNA has been
probed using ribonucleases and DEPC, and the data largely support the
secondary structure predictions shown in Fig. 1. However, the internal
loop region of the RNA ligand, shown in Fig. 1 as single-stranded, is
clearly structured in some way. Binding interference studies and the
use of sequence variants of the RNA have demonstrated that this
structured internal loop, and the regions immediately flanking it, are
the critical elements in tight binding to the MetJ repressor.
The exact details of the interaction between the two macromolecules
will require x-ray or NMR structural information, and the experiments
described here have helped to define a minimal RNA target for
structural studies. The most likely binding location for the SELEX RNA
ligand is the DNA binding site. MetJ inserts two Many protein-nucleic acid complexes are stabilized by large numbers of
contacts to the phosphate backbone. Thus each MetJ dimer contributes 19 direct hydrogen bonds to phosphate groups when binding DNA (4).
However, with the MetJ-RNA interaction, protein-phosphate contacts seem
to play, at most, a very minor role. Phosphate ethylation failed to
identify important phosphate groups for the interaction, implying that
charge neutralization is not a major component of the RNA-protein
affinity. Similar weak ionic dependences have been observed for
physiologically important RNA-protein interactions, such as the binding
of ribosomal protein L11 to its target within 23 S rRNA (15).
In summary it is clear that MetJ is capable of tight and specific
binding to its natural DNA target, the Met box, and also to a selected
RNA ligand. The nature of the interaction between the protein and these
two nucleic acids is, however, very different. With DNA there are only
a few interactions with the bases and multiple contacts to phosphates.
With RNA interactions the bases dominate and the phosphates play only a
minor role. The RNA aptamer therefore appears to be a novel solution to
the MetJ recognition problem.
INTRODUCTION
Top
Abstract
Introduction
References
topology where the
-strands from each
subunit intertwine to form an anti-parallel
-ribbon, which lies in
the major groove of the target DNA. Binding specificity is largely
determined by hydrogen bonds between the side chains of lysine 23 and
threonine 25 of the
-strands and 4 purine bases in each Met box.
Many hydrogen bonds are also made between the protein and the DNA
phosphate backbone, and there is evidence that the conformation of the
backbone is also important for binding (4). Binding of the MetJ
repressor to DNA is modulated by S-adenosylmethionine (AdoMet),1 which markedly
increases the affinity of MetJ for its target sequence. AdoMet and the
DNA are bound on opposite faces of the protein, and binding of AdoMet
does not significantly perturb the protein structure. The increased
affinity of the holorepressor appears to be due to a long range
electrostatic effect caused by the positively charged tertiary sulfur
atom of AdoMet (5, 6).
EXPERIMENTAL PROCEDURES
20 °C. Base
composition analysis was carried out as described previously (20), and
the results were consistent with the predicted values for the oligoribonucleotide.
-32P]ATP (3000 Ci/mmol; Amersham) and
T4 polynucleotide kinase (Amersham Pharmacia Biotech) as described
previously (19). Alternatively, the 3'-ends were labeled by the
ligation of [5'-32P]pCp (3000 Ci/mmol; Amersham Pharmacia
Biotech) to the 3'-end using RNA ligase (Amersham Pharmacia Biotech)
(21). Labeled RNA was purified on 1.5-mm 15% (w/v) polyacrylamide gels
containing 7 M urea. RNA was eluted from the appropriate
gel fragment by incubation in 0.1 M Tris/HCl, pH 8, 100 mM NaCl, 1 mM EDTA, 0.1% (w/v) SDS for 12 h at 37 °C. RNA was precipitated by the addition of 3 volumes of
absolute ethanol. The pellet was collected by centrifugation, rinsed
with absolute ethanol, and dried, before being resuspended in 50 µl
of sterile water.
1 pg,
1 × 106 cpm) was digested with RNase
T1 (Boehringer Mannheim), RNase A (Sigma), or RNase
U2 (Sigma) in 20 µl of 50 mM Tris/HCl, pH
7.0, 50 mM KCl, containing 6 units of tRNA (type X-SA,
Sigma). Digestions were carried out at 37 °C for 15 min. The cleaved
products were analyzed on a denaturing 19% (w/v) polyacrylamide gel
containing 7 M urea and visualized by autoradiography or
phosphorimaging (Fujifilm BAS 1500). Quantitation was carried out using
the PhosphorImager associated software, TINA (Raytest
Isotopenme
geräte GmbH).
5 × 106 cpm) of 5'- or 3'-labeled RNA in either
200 µl of native buffer (50 mM Tris/HCl, pH 7.0, 50 mM KCl) or 200 µl of denaturing buffer (50 mM
sodium acetate, pH 4.5, 1 mM EDTA) were modified with 5 µl of DEPC at either 37 °C for 30 min (native buffer) or 90 °C for 2.5 min (denaturing buffer). The reaction was stopped with 75 µl
of 1 M sodium acetate, pH 4.5, and 750 µl of absolute
ethanol. Following centrifugation the RNA pellet was dissolved in 200 µl of 0.3 M sodium acetate, pH 3.8, and precipitated with
600 µl of absolute ethanol. The precipitated RNA was rinsed with
absolute ethanol and dried. DEPC-modified RNA was then cleaved at the
modified nucleotide by treatment with aniline (24). The fragmented
products were examined by denaturing gel electrophoresis, as above.
6 pg,
5 × 106 cpm) in 10 µl of hydrazine hydrate (24).
Following incubation on ice for 10 min, the reaction was stopped by the
addition of 200 µl of 0.3 M sodium acetate, pH 3.8, and
750 µl of absolute ethanol. The RNA pellet was re-precipitated, as
described above, and dissolved in 15 µl of water. RNA phosphate
ethylation was carried out with ethylnitrosourea (25). About 6 pg of
labeled RNA (
5 × 106 cpm) in 0.3 M
sodium cacodylate, pH 8.0, 2 mM EDTA, was treated with 4 µl of ethylnitrosourea (as a saturated ethanolic solution) at
80 °C for 2 min. The reaction was stopped by the addition of 3 µl
of 3 M sodium acetate, pH 6.0, and 100 µl of absolute
ethanol. After centrifugation, the RNA pellet was resuspended in 20 µl of 0.3 M sodium acetate, pH 6.0, 20 mM
EDTA, and reprecipitated with 100 µl of absolute ethanol. The RNA
pellet was rinsed with absolute ethanol, dried, and resuspended in 15 µl of water.
RESULTS AND DISCUSSION
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Fig. 1.
A, the sequence and predicted secondary
structure of the RNA ligand used in this study. B and
C, the truncated RNA ligands, in which parts of the sequence
shown in A were replaced with a hexaethylene glycol
linker.
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Fig. 2.
A, enzymatic probing of the secondary
structure of the RNA ligand. RNA, labeled at either the 5' or 3' end,
was digested with ribonucleases specific for G (RNase T1),
A (RNase U2), or C/U (RNase A). B, chemical
probing of the secondary structure of the RNA ligand. RNA, labeled at
either the 5' or 3' end, was modified in either native (N)
(50 mM Tris/HCl, pH 7.0, 50 mM KCl; 37 °C)
or denaturing (D) (50 mM sodium acetate, pH 4.5, 1 mM EDTA; 90 °C) buffer with DEPC and then subjected to
aniline cleavage. Following enzymatic or chemical treatment, the
ladders obtained were separated by gel electrophoresis and detected by
autoradiography or using a PhosphorImager. The RNA sequence
(A) or the A and G bases modified (B) are
indicated at the side of the gels.
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Fig. 3.
Summary of secondary structure mapping of the
RNA ligand. A, sensitivity of individual bases in the
RNA to RNase U2 (A-specific) and RNase A (C/U-specific) is
indicated by circles and triangles, respectively.
Three symbols represent high sensitivity to the nuclease;
two, intermediate sensitivity; one, moderate
sensitivity. B, purines that are modified by DEPC in a
denaturing, but not a native, buffer are shown with circles.
Three symbols represent a strong difference in
DEPC sensitivity and one symbol a moderate
difference.
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Fig. 4.
Interference analysis of the interaction
between the RNA ligand and MetJ using DEPC modification. Both 5'-
and 3'-labeled RNA was used. Following DEPC treatment, and incubation
with MetJ, the free and bound RNA fractions were separated by gel-shift
electrophoresis, recovered, and then cleaved with aniline. The ladders
obtained were analyzed by electrophoresis and detected by
autoradiography or using a PhosphorImager. a, DEPC-treated
RNA that was gel-shifted by MetJ protein; b, DEPC-treated
RNA that failed to gel-shift in the presence of MetJ protein;
c, DEPC-treated RNA run on the gel in the absence of
protein. Positions of the purines are shown at the side of the
gel.
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Fig. 5.
Densitometry of the interference data found
for MetJ using DEPC and hydrazine-modified RNA. A,
3'-labeled RNA modified with DEPC; B, 5'-labeled RNA
modified with DEPC; C, 3'-labeled RNA modified with
hydrazine; D, 5'-labeled RNA modified with hydrazine.
Black, RNA that was gel-shifted by MetJ; red, RNA
that failed to shift in the presence of MetJ protein; blue,
RNA run on the gel in the absence of protein.
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Fig. 6.
Summary of the interference analysis data
found for MetJ using DEPC and hydrazine-modified RNA. Interference
by DEPC and hydrazine are denoted by circles and
triangles, respectively. Two symbols
represent strong interference and one symbol
signifies interference. Strong interference and interference are
defined by modification interference indexes of greater than 4 and
between 2 and 4, respectively (14).
7
M were seen for both the deletions and the parent RNA
ligand. A control RNA, consisting of the first 21 nucleotides of the
parent RNA, did not bind to MetJ. These results clearly demonstrate
that the initial, synthetic SELEX RNA fragment used does in fact bind tightly to MetJ as expected from the original SELEX
experiment.2 Furthermore, we confirm that the internal loop
of the RNA ligand encompasses the high affinity site for the MetJ
repressor, in agreement with the binding-interference analysis.
Although the terminal loop is not directly involved in binding it is
necessary to allow the reversal of the RNA strand and hence the
formation of the internal loop.
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Fig. 7.
The interaction of RNA ligands with MetJ as
assessed by filter binding. ------
, intact RNA
(structure A in Fig. 1);
- - -
, RNA
with 6 bases replaced by a hexaethylene glycol spacer
(structure B in Fig. 1);
····
, RNA
with 16 bases replaced by a hexaethylene glycol spacer
(structure C in Fig. 1). The line along the
bottom of the graph (solid squares) is a control
RNA consisting of nucleotides 1-21 (top half) of the intact RNA. The
individual data points shown are the average of three readings. The
error in the averaged measurement was between 5 and 10%. Each binding
isotherm was determined four times to give Kd
values.
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Fig. 8.
Densitometry of the interference data found
for MetJ using ethylnitrosourea to ethylate RNA phosphates.
A, 5'-labeled RNA; B, 3'-labeled RNA. ------, RNA
that was gel-shifted by MetJ protein; ····, RNA that failed to
shift in the presence of MetJ protein. 19/18 is the
phosphate between bases 18 and 19 in Fig. 1, etc. Some peaks are
indicated merely for the purpose of identification.
7 M were obtained. The dissociation
constant was marginally higher at KCl concentrations of 150 and 200 mM, 2.3 ± 0.5 × 10
7 and 6.3 ± 1 × 10
7 M, respectively.
Interestingly, although the data at 100-200 mM salt fitted
a typical protein-ligand binding isotherm, those at 10-50
mM KCl were notably steeper, suggesting that some form of
cooperativity occurs. One possible explanation is that MetJ dimers may
dissociate at low concentrations in low salt buffers. Although it has
been shown that thermally denatured MetJ protein will readily
reassociate (30), we have no evidence for this dissociation. However,
the related Arc repressor from bacteriophage P22 does dissociate into
unfolded monomers with a dissociation constant of 5 × 10
9 M (31, 32). Alternatively,
salt-dependent conformational changes of the RNA ligand
could be involved, although the salt concentrations examined here had
no effect upon the circular dichroism spectra of the RNA (not shown).
Steep binding curves have been observed for the binding of MetJ protein
to its DNA target (3). Because of these complexities caution is needed
in the interpretation of how the Kd varies with
ionic strength. Nevertheless, the minor effect of salt concentration
upon the affinity of the RNA ligand binding to MetJ agrees with the
lack of phosphate ethylation interference. Both experiments suggest
that interactions between the phosphates of the RNA ligand and the MetJ
play a minor role in binding.
at 263 nm and a small
negative peak at 295 nm, typical of an A-form helix. Heating the RNA
caused a decrease in the intensity of the main peak and a shift to 271 nm, consistent with the conversion of the RNA to a single strand (33).
This is consistent with the mapping data presented above, which show
that the internal loop cannot be considered single-stranded, but must
have some element of structure. Thus, with the exception of the
single-stranded terminal loop, almost all of the RNA ligand appears to
exist as an A-form duplex. However, the exact structure of the internal loop may deviate somewhat from classical A-duplex parameters. When the
RNA ligand binds to the MetJ protein, there is hardly any change in the
circular dichroism spectrum of the nucleic acid (Fig. 9), especially
above 240 nm where almost all (
90%) of the intensity is due to the
nucleic acid. This suggests that there are no large conformational
changes to the RNA after binding to the protein.
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Fig. 9.
Circular dichroism spectra of the RNA.
A, circular dichroism spectra of the RNA containing 46 nucleotides (Fig. 1A) at 37 °C (solid
line) and at 90 °C (dashed line).
B, spectra of RNA with increasing concentrations of MetJ.
RNA alone, solid line; 5.5 × 10 7 M MetJ (dimer), dotted
line; 5.5 × 10
6 M Met J
(dimer), dashed line. The spectrum of the free
protein was subtracted from the last two spectra. In each case the RNA
concentration was 10
6 M. Above 240 nm the
protein spectrum was
10% the intensity of that of the RNA giving a
correction of
10%. Below 240 nm the intensities of both spectra
became comparable and so accurate subtraction was not possible. This
probably accounts for any deviation between the three spectra below 240 nm.
CONCLUSION
-strands into the
major groove of its DNA target, and this element is used to generate
specificity. Although the large major groove of B-DNA can accommodate
the
-ribbon, it would not fit into the smaller major groove of
A-form double-stranded RNA. It is possible that the internal loop forms
a loose, double-stranded structure, with either the major or minor
grooves being large enough for binding of the
-ribbon. The
nucleotides in and directly adjacent to the internal loop were found to
be highly conserved during the SELEX procedure,2 consistent
with the important role of this region in tight binding. Indeed, the
degree of conservation is surprisingly high, and a few minor
differences, such as single nucleotide substitutions, might be
expected. With the Met box DNA recognition sequence a number of
substitutions can be readily accommodated, and only a small number of
bases from each strand are directly contacted (4). We propose that
most, if not all, of the bases in the internal loop of the RNA ligand
play critical roles either by making hydrogen bond contacts to MetJ or
maintaining structural integrity. Thus many more contacts are likely to
be made between the MetJ and the RNA bases compared with the bases of
the DNA operator. The finding that the RNA ligand undergoes little
conformational change on binding to MetJ contrasts with many other
examples where protein binding results in significant conformational
changes to the RNA ligands (36, 37).
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FOOTNOTES |
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* This work was supported by a grant from the Wellcome Trust (United Kingdom) (to B. A. C. and P. G. S.) and by grants from the UK BBSRC and the University of Leeds (to P. G. S.).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.
¶ Current address: Dept. of Chemistry, Indiana University, Bloomington, IN 47405.
To whom correspondence should be addressed. Tel.:
44-191-222-7371; Fax: 44-191-222-7424; E-mail:
b.a.connolly{at}ncl.ac.uk.
The abbreviations used are: AdoMet, S-adenosylmethionine; HIV, human immunodeficiency virus; SELEX, systematic evolution of ligands by exponential enrichment; DEPC, diethyl pyrocarbonate.
2 Y.-Y. He, A. L. Ellison, I. D. Parsons, P. G. Stockley, and L. Gold, manuscript in preparation.
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REFERENCES |
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