From the Department of Pharmacology, University of Minnesota, Minneapolis, Minnesota 55455-0347
Received for publication, November 13, 2000, and in revised form, February 5, 2001
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ABSTRACT |
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Vertebrates control intracellular iron
concentration principally through the interaction of iron regulatory
proteins with mRNAs that contain an iron responsive element, a
small hairpin with a bulged C. The hairpin loop and bulged C have
previously been assumed to be critical for binding and have been
proposed to make direct contact with the iron regulatory proteins.
However, we show here that a U or G can be substituted for the bulged C provided that specific nucleotides are also present within internal loops. The Kd, IC50 and chemical
modifications of the iron responsive element variants are similar to
the wild-type. Results are more consistent with a role in which the
C-bulge functions to orient the hairpin for optimal protein binding
rather than to directly contact the protein. Characterization of
these novel iron responsive element variants may facilitate the
identification of additional mRNAs whose expression is controlled
by iron regulatory proteins, as well as provide insight into the nature
of a critical RNA-protein interaction.
A decrease in cytosolic iron concentration can stimulate the RNA
binding activity of two different iron regulatory proteins (IRP-1 and
IRP-2).1 IRP-1 and
cytoplasmic aconitase are the same protein, but the RNA binding and
enzymatic activities are mutually exclusive (1-4). When the
intracellular iron concentration decreases, an iron-sulfur cluster
necessary for aconitase activity is disassembled, which then activates
the RNA binding site. IRP-2 has 57% overall amino acid identity
to IRP-1 but contains an additional 73-amino acid insertion and is not
converted to an inactive form under iron-replete conditions (5-7).
Instead, interaction of aqueous iron within the 73-amino acid insertion
results in oxidation of the protein and targeting for
ubiquitin-dependent degradation (8, 9). The RNA binding of
both proteins can also be affected by nitric oxide, oxidative stress,
and phosphorylation (reviewed in Refs. 10, 11).
Intracellular iron importation, storage, and utilization are largely
regulated through interactions of the IRPs with iron responsive
elements (IREs) that are present on several mRNAs (12-20). The
mRNA encoding the transferrin receptor, the major means of iron
importation for most mammalian cells, has five IREs within its
3'-untranslated region. The binding of an IRP to this structure masks a
ribonuclease site which increases the half-life of the message and
leads to increased iron importation (12, 13). The mRNAs encoding
the H and L ferritin subunits, the major iron storage proteins of the
cell, both have an IRE near the 5'-end (14, 15). IRP binding inhibits
translation by preventing recruitment of the small ribosomal subunit to
these mRNAs, which leads to decreased iron storage (16). As a
result, IRP binding to the transferrin and ferritin mRNAs can
function synergistically to elevate the cytosolic iron concentration.
The expression of the citric acid cycle enzyme mitochondrial aconitase
and the heme biosynthetic enzyme erythroid Mutagenesis (21-24), phylogenetic analysis (25), in vitro
selection (26, 27), chemical probing (28, 29), and NMR (30-32) have
defined structural features of the IRE, and there is significant
overlap in the structures recognized by both IRPs. The natural IREs all
contain the hairpin loop sequence CAGWGH (where W is A or U and H is C,
A, or U; Fig. 1A) that is necessary for high affinity
binding to both IRPs. NMR and in vitro selection results
indicate that the first and fifth positions of the hairpin loop form a
Watson-Crick pair. A disordered C nucleotide is present in all
identified natural IREs five base pairs from the hairpin loop (Fig.
1A, C-12) and has been assumed to directly contact the IRPs.
In most IREs, the C is a single bulge in the helix of the IRE stem
(32), but it is also found as part of a larger dynamic internal loop
(31). This internal loop increases binding affinity for the IRPs and
has been proposed to be part of a Mg2+ binding site that
could affect the conformation of the hairpin (33). Our results indicate
that a U or G can substitute for the C with little effect on binding to
IRP-1 provided that additional nucleotides are also present within the
bulged loops. Results are more consistent with a role for the IRE
bulge/bulge loop in positioning the hairpin for optimal IRP-1 binding
rather than in directly contacting the protein.
Protein Preparation--
Human IRP-1 with an N-terminal
His6 tag was expressed in Escherichia coli as
previously described (34) and purified by Q-Sepharose and
nickel-chelate chromatography followed by dialysis against 25 mM Hepes, pH 7.6, 150 mM KOAc, and 1.5 mM MgCl2 to remove the imidazole. The protein
was 98% pure as estimated from silver staining. Glycerol was added to
a final concentration of 5% prior to freezing.
RNA Preparation--
The starting random RNA population
was synthesized by T7 transcription from the double stranded DNA
template:
CGGAAGCTTCTGCTACATGCAATGG(N)50CACGTGTAGTATCCTCTCCCTATA GTGAGTCGTATTA, where N in theory is an equal representation of each of
the four standard nucleotides. For the chemical modification studies,
the selected U-bulge RNA (Fig. 1B) was synthesized by T7
transcription from a DNA template that added a 3' extension (AUAUUACGAGUAUAUGGUGUAGG). This extension has less than a 2-fold effect
on binding affinity (data not shown) and was used as a primer binding
site for the detection of the modifications. All other RNAs were
synthesized by T7 transcription from the corresponding oligodeoxynucleotide template.
Selections--
The initial cycle of selection contained 200 pmol of the 50N random RNA. Assuming no other biases, this would have
resulted in a 99% probability of having representation of any
combination of 22 contiguous nucleotides (35). The RNA was incubated
with 20 pmol of purified IRP-1 in 100 µl of 1× native buffer (10 mM Hepes, pH 7.5, 3 mM MgCl2, 40 mM KCl, 50 ng/µl bovine serum albumin, and 1%
2-mercaptoethanol). The binding reaction for the first 9 cycles was
done at 22 °C. This was increased to 37 °C for the last four
cycles to remove less stable structures. For cycles 1 to 7 and 10 to
13, the bound RNA was partitioned from the bulk population by
filtration through nitrocellulose. The bound complex was partitioned on
a native polyacrylamide gel for cycles 8 and 9 to decrease the
likelihood that RNAs would be selected for an unintended criterion. In
addition, a negative selection was exploited before cycles 2 through 6 so as to remove RNAs within the population with affinity to
nitrocellulose or minor contaminants of the IRP-1 preparation. A
100-fold molar excess of the ferritin IRE (Fig. 1A) was
included in the binding reaction of the 9th cycle of selection to
compete away lower affinity RNAs. The selected U-bulge RNA was
randomized at eight positions and subjected to three rounds of
re-selection. The IRP·RNA complex was partitioned by filtration through nitrocellulose for the first 2 cycles and by native gel electrophoresis for the third. A 100-fold molar excess of the ferritin
IRE was also included in the binding reaction of the third cycle of
re-selection.
A Bulged C Is Not Essential--
We initially exploited in
vitro selection (36, 37) to generate novel high affinity IRP-1
binding RNAs from a starting population that had 50 random positions
(50N). Unlike previous selections (26, 27), the random region was large
enough to permit the selection of alternative structures requiring more sequence space, and in addition was designed to select for the highest
affinity interactions (38). RNAs were cloned and sequenced after both
the 9th and 13th cycles of enrichment. The sequence phylogeny and IRP-1
binding affinities are similar for both RNA populations (data not
shown). All of the 27 sequenced clones contain the IRE hairpin loop
(CAGWGH) confirming the importance of this sequence. However, two of
the selected high affinity RNAs have a U or a G at the bulge position
rather than a C.
Binding affinity of the IRE variants for IRP-1 was determined by two
methods. First, binding curves were generated by incubating the
radiolabeled RNA with increasing quantities of purified IRP-1 and
partitioning the bound complexes from the free RNA by filtration through nitrocellulose. The Kd was determined from
an analysis of the binding curve that did not require an assumption that [IRP]free ~ [IRP]total (39). The
measured value of 40 ± 30 pM (n = 3)
for the affinity of the ferritin IRE is in agreement with previously
published values (24, 40). The second method determined the
Kd relative to that of a human ferritin IRE using a
competition assay (Fig. 1C).
Radiolabeled ferritin IRE RNA was incubated with IRP-1 in the presence
of increasing quantities of unlabeled RNA competitor. A value for
Krel is defined as the IC50 of the
unlabeled competitor RNA normalized to the concentration of the
radiolabeled ferritin IRE. All data points for the binding curves were
done in triplicate and the R2 for the curve fits
is >0.9 for those variants that have a Kd or
Krel value within a factor of 20 of the
wild-type ferritin IRE and >0.8 for the other variants. The
Kd or Krel values for some
RNAs were obtained from several independent curves, and the means and
S.D. are indicated (Fig. 1).
The selected RNAs were folded in order to be most consistent with the
chemical modification data (Fig. 2) as
well as with the folding of the ferritin IRE, for which there is a
larger body of supporting data. An RNA selected after 9 cycles of
enrichment has a U at the bulge position and also differs significantly
from the wild-type ferritin IRE in that there are two extra nucleotides at the 3'-end of the hairpin loop (CA), one additional C nucleotide within the top bulge and two within the bottom bulge (Fig.
1B). The PCR primer binding sequences were deleted from the
selected RNA, and the lower stem was stabilized with two additional
G-C pairs. The affinity of IRP-1 for this RNA, measured both directly or by competition (Krel), is ~50% that of the
wild-type ferritin IRE. In the absence of the additional CA, the
selected U-bulge motif has binding affinity equal to that of the
wild-type ferritin sequence (Krel = 0.97 ± 0.02, n = 2) suggesting that the CA not only does not
contribute binding energy but is detrimental. This is supported by the
observation that the addition of the extra CA to the hairpin loop of
the ferritin IRE also decreased binding affinity by 2-fold (Fig.
1A).
Mutation of the bulged C in the wild-type ferritin IRE to a U decreased
binding affinity by almost an order of magnitude, in agreement with
published results (Fig. 1A; Refs. 23, 27). To determine why
a U at the bulge position of the selected RNA does not likewise inhibit
binding, a series of mutations were made. Mutations to the top stem of
the selected RNA (nt 11-15 and 24-28 in Fig. 1B) had no
effect on binding affinity provided that Watson-Crick pairing was
maintained, which was demonstrated for the ferritin IRE (21). Deletion
of the additional C nucleotides from the bulge region of the selected
RNA (nt 7 or 29 and 30) dramatically inhibited IRP-1 binding. The
importance of the conformation of this region is emphasized by the
significant inhibition caused by the addition of an A between
nucleotides 31 and 32 (Krel >40, n = 3). The corresponding addition to the ferritin IRE
also inhibited IRP binding, although not as dramatically (Fig.
1A). For both RNAs, the added nucleotide potentially
stabilizes the lower helix. In the context of the ferritin IRE, the
inserted A could completely pair the lower stem as is the case for the
transferrin receptor type IRE, which also has a lower affinity for the
IRPs (41).
The U-A and C-G pairs of the lower ferritin stem (bp 7-32 and 8-31
in Fig. 1A) and the U-G and G-C pairs adjacent to the
bulged C (bp 10-30 and 11-29) can also form in the selected U-bulge
RNA. The significance of this sequence to the binding affinity of the ferritin IRE has not been fully appreciated. However, its presence in
the selected U-bulge RNA emphasizes the contribution of this region.
The U-A and C-G pairs in the lower stem of the selected RNA
contribute base-specific binding affinity as indicated by the
substitutions that maintain Watson-Crick pairing and cause a 2-fold
increase in Krel (bp 4-34 and 5-33 in Fig.
1B). The corresponding nucleotides in the ferritin IRE have
previously been suggested to be part of a Mg2+ binding site
that optimizes IRP binding (31). The mutagenesis indicates, therefore,
that the additional bulged nucleotides and the presumed
Mg2+ binding site of the selected U-bulge RNA are necessary
to compensate for the lack of a C at the bulge position.
Chemical Probing--
To obtain support for the proposed structure
of the selected U-bulge RNA, the molecule was probed with
dimethylsulfate (DMS) and 1-cyclohexyl-3-(2-morpholinoethyl)
carbodiimide metho-p-toluene sulfonate (CMCT) under
denaturing and native conditions (Ref. 42; Fig. 2A). Primer
extension analysis was used to detect DMS modifications at N1-A and
N3-C and the CMCT modifications at N3-U and N1-G (Fig. 2A).
These functional groups would be protected from modification by
secondary or tertiary interactions and by protein contacts. The
detected modifications were consistent with the proposed structure with
two exceptions (Fig. 2C). First, no evidence was obtained
for the intraloop base pair (16C-G20), however
the dynamic nature of a single base pair may not be sufficient to
provide protection. Second, C-7 is predicted to be bulged yet is
protected from modification under native conditions. This protection
could have resulted either from a tertiary interaction or alternatively
the model of the secondary structure of the bulge region may not be
completely correct (Fig. 1B). In the NMR structure of the
frog ferritin IRE, the G corresponding to G-32 of the selected U-bulge
RNA is base-paired in alternative conformations to either a U
corresponding to U-8 or a U that is at the position corresponding to
C-7 of the selected U-bulge RNA (31). The chemical probing is more
consistent with this dynamic model. Structural information for C-5 and
C-6 could not be obtained as neither base is reactive under denaturing
conditions, and the evaluation of the reactivity of A-14, C-15, C-16,
C-35, and C-36 is not possible because of modification-independent RT terminations.
The hairpin loop nucleotides A-17, G-18, U-19, G-20, and U-21 are
protected from modification by IRP-1 consistent with the protection of
the transferrin receptor IREs (Fig. 2A, lanes 8 and
12; Ref. 28). The entire ferritin IRE has previously been reported to be protected by IRPs when probed with enzymes and metal
coordination complexes (43, 44). However, this discrepancy probably
reflects the properties of the different probes and as a result, the
two sets of protections are not necessarily inconsistent. IRP-1 also
protects U-8 and U-10 from CMCT modification. These protections could
have resulted either from a direct contact with the protein or
alternatively because the protein induces a conformational change
within the RNA. The extra loop nucleotides C-22 and A-23 are not
protected, suggesting that they are not making direct contact with the
protein, in agreement with the mutagenesis.
The selected U-bulge RNA was also probed with a
1,10-phenanthroline-copper complex (OP-Cu), which cleaves
single-stranded regions of RNA (45). Sites of cleavage were mapped by
primer extension (Fig. 2B). There are three principal
cleaved regions: the bottom and top bulges as well as the 3'-end of the
hairpin loop (Fig. 2C). These regions of the ferritin IRE
were previously shown to be cleaved by OP-Cu, and the cleavage was
sensitive to the Mg2+ concentration (29). Titration of
Mg2+ also inhibits the OP-Cu-dependent cleavage
of the U-bulge RNA. In both the ferritin and the U-bulge RNAs, the
Mg2+ could be competing for the same binding site(s) as the
OP-Cu complex. Because the effect of the Mg2+ titration is
similar for all three regions, it is possible that the bulge loops and
hairpin loop could be forming a single Mg2+/OP-Cu binding
site that functions to optimize IRP-1 binding. Alternatively, there
could be a binding site for Mg2+, which when occupied
dramatically alters the conformation of the IRE, rendering all of the
sites inaccessible to OP-Cu cleavage.
Internal Loop Sequence Requirement--
Because the mutagenesis
clearly indicates that the internal loops of the selected U-bulge RNA
are critical for IRP binding, we were interested in a more detailed
analysis of the sequence requirements of this region. Only one RNA was
initially selected with a U at the bulge position, possibly because the
requirement of the internal loop nucleotides resulted in less
representation within the starting random RNA population. To generate a
phylogeny with which to obtain additional structural information, three nucleotides in the top bulge loop (nt 7-9 in Fig.
3A) and three in the bottom
(nt 29-31) were made random and were reselected for high affinity
binding to IRP-1. The additional CA (nt 22-23) of the hairpin loop was
also randomized for the reselection. The measured values for the
Krel of the reselected clones are within a
factor of three of the parental RNA (Fig. 3B). The parental top and bottom loop sequences were reselected (Fig. 3B, clone 12), and no RNA was selected with an A at position 7, 8, or 31 or
a G at position 30, emphasizing a sequence preference in the internal
loops. All five of the clones that were selected with a G at position 8 also have a C at position 30. Furthermore, two additional clones with a
U at position 8 also have an A at position 30. This co-variation would
suggest that these two positions interact. However, the remaining seven
reselected clones and the parental sequence would either have a U-C or
C-U non-Watson-Crick pair at these positions, suggesting either that
these particular mismatches were tolerated or that the RNAs form an
alternative structure in this region more consistent with that in Fig.
1B. A significant degree of variability is tolerated at
positions 22 and 23 of the hairpin loop, but only one RNA was selected
with a G at either position possibly because of the potential to
compete with G-20 for the formation of the intraloop base pair with
C-16 (Fig. 3A).
An IRE variant was also obtained from the 13th cycle of the original
50N selection with a G at the bulge position (Fig. 3C). The
PCR primer binding sequences were deleted from the RNA, and the lower
stem was stabilized with two additional G-C base pairs. The
Kd and Krel for the binding
of this RNA to IRP-1 indicates the affinity for IRP-1 is approximately
the same as the wild-type ferritin IRE, yet substitution of the C with
a G at the bulge position of the ferritin IRE results in a 6-fold decrease in affinity, consistent with previously published results (Fig. 1A; Refs. 23, 27). The G-bulge RNA, like the U-bulge, contains additional internal loop nucleotides. Clone 11 (Fig. 3B) from the reselection of the U-bulge RNA has the same
internal loop sequence as that found in the G-bulge RNA, suggesting
that these nucleotides perform the same function in both RNAs.
mRNAs Containing the IRE Variants--
The identification of
the novel high affinity IRE variants lacking a bulged C enabled
expanded searches of DNA data banks for additional mRNAs whose
expression could be regulated by IRPs. A human EST data bank was
searched using an algorithm that exploits RNA secondary structure
constraints (46).2 ESTs were
identified that have the potential to form the CAGWGH hairpin loop five
base pairs removed from a C, G, or U that is present as part of a bulge
or internal loop. Because an IRE needs to be within 100 nucleotides of
the mRNA 5'-end to regulate translation (16), only the identified
ESTs encoding mRNAs with a mapped 5'-end could be evaluated for
biological relevancy. We initially identified 696 unique ESTs that have
the potential to form an IRE structure within 100 nucleotides of the
EST 5'-end.3 Of these, 105 encode an annotated mRNA with a mapped 5'-end, and seven of the
mRNAs potentially can form an IRE-like structure within 100 nucleotides of its 5'-end. However, the bulged loop sequences of the
seven putative IREs are not identical to those described here (Fig.
3B), and the measured Kd of these RNAs
for IRP-1 is higher than that expected for biological relevancy. The
remaining 591 non-annotated ESTs could not be readily evaluated for
biological relevancy, but as the genome continues to be characterized, the novel U and G bulge variants may facilitate the identification of
additional IRP-regulated mRNAs.
The absolute conservation of the CAGWGH hairpin loop within the
RNAs selected from the starting 50N RNA population emphasizes the
importance of this sequence, and it is remarkable that more highly
divergent RNAs were not obtained given the available sequence space.
However, the selection was set-up to isolate the highest affinity IRP-1
binders, and as a result RNAs with dramatically different conformations
and lower binding affinities, analogous to those that interact with the
E. coli aconitase (47), would not have been selected. In
addition, high affinity binding RNAs requiring significantly more
sequence space than the IRE may have been underrepresented within the
starting RNA pool. Other unintended biases such as the impact of the
flanking PCR primer sequences also have to be considered, and as a
result the possibility of additional high affinity IRP-binding RNA
motifs cannot be excluded. Alternatively, the binding affinity of the
natural IRE to an IRP is one of the strongest known RNA-protein
interactions, and the CAGWGH hairpin loop may simply be an integral
part of the best possible IRP binding RNAs.
The in vitro selections and mutagenesis indicated that the
addition of a CA to the 3'-end of the hairpin loop only results in a
2-fold decrease in binding of the ferritin IRE and selected U-bulge RNA
(Fig. 1). Previous insertions to the hairpin loop had a significantly
greater detrimental effect (23, 48). However, these RNAs had
nucleotides added to both the 5'- and 3'-sides of the hairpin loop, and
it is likely that the location and size of the insertion as well as the
sequence bias against G (Fig. 3B) affects the relative
impact of the insertions. This is supported by the absence of
insertions at other locations within the hairpin loop of the RNAs
obtained from the initial 50N selection.
On the basis of limited in vitro selection and mutagenesis,
it had previously been assumed that a bulged C was essential for high
affinity binding of the IRE to the IRPs. The initial direction of this
study used IRP-1 to test the hypothesis that there are additional high
affinity variants of the IRE that could not have been detected by the
previous studies. The internal bulges/loops that can compensate for a U
or G at the bulge position make this point. Putative IREs with a G or U
at the bulge position have also been identified within the crayfish
ferritin mRNA (49) and the trout ferritin mRNA (50)
respectively. Both putative IREs also have the potential to form
internal loops although not with the same sequence as that described
here (Fig. 3B), and their physiological relevance has not
yet been demonstrated. IRP-2 has previously been shown to be more
sensitive to the presence of the internal loops than IRP-1 (41). As a
result, even though IRP-1 and IRP-2 are highly similar proteins with
overlapping and similar binding affinities, it is possible that IRP-2
may not interact with the selected RNAs in the same manner as
IRP-1.
There are at least two mechanisms through which the internal
loops could be facilitating the binding of the IRE variants containing a U or G substitution at the bulge position. First, the selected loops
could provide binding energy by making direct contact with IRP-1, in
effect compensating for the loss of contacts potentially provided by a
bulged C. However, the tolerated sequence heterogeneity of the selected
bulges/loops (Fig. 3B) would argue against direct protein
contacts. In addition, conservation of the CAGWGH hairpin loop, the
similar IC50 (Krel) and
Kd, and chemical protections would argue against
significant differences in the binding of the selected RNAs to IRP-1.
Also, substitution of U-10 with a C in the selected U-bulge RNA does
not significantly improve binding, which may have been expected if the
selected loops were making additional contacts (Fig. 1B).
Alternatively, if the role of the ferritin IRE bulged C is to position
the phylogenetically conserved hairpin loop for optimal binding, the
selected loop nucleotides may provide another means of obtaining this
conformation when there is a U or G at the bulge position. This is
consistent with NMR results suggesting the internal loop influences the
conformation of the hairpin loop (33, 41). However, because the loss of one or more potential hydrogen bond contacts may only have a slight impact on Kd, the possibility that the bulged C of
the ferritin IRE also makes some minor contacts with the protein cannot be completely excluded. Aconitase purified from Bacillus
subtilis binds to a B. subtilis mRNA that has the
potential to form an IRE-like structure containing the CAGWGH loop
sequence but missing the canonical bulged C (51). This further suggests
that the hairpin loop sequence is the central feature of the IRE-IRP
interaction and implies that the internal bulge could have subsequently
evolved to optimize the interaction.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-aminolevulinate synthase
are also regulated through IRE-IRP interactions (17, 18). In addition, putative IREs have also been identified on the mRNAs encoding the
iron transporters DMT1 and IREG1 (19, 20). Characterization of the
IRE-IRP interaction, therefore, is of high biological significance.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
The C-bulge is not essential for high
affinity IRE binding. A, secondary structure of the
human ferritin H-chain IRE and the IRP-1 binding affinity of several
mutations. Lowercase letters are not part of the natural IRE
sequence and have little effect upon IRP-1 binding. B,
predicted secondary structure of the selected U-bulge RNA and the
binding affinity of several mutations. The U-10 to C substitution (*)
was made in the context of an RNA lacking C-22 and A-23. All other
mutations were made in the context of the indicated parental RNA.
C, representative competition curves. Radiolabeled ferritin
IRE was incubated with IRP-1 in the presence of increasing
concentrations of the unlabeled ferritin IRE, the selected U-bulge RNA
and the U-bulge RNA with the CA deleted from the hairpin.
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Fig. 2.
Chemical probing of the selected U-bulge
RNA. RT terminations that are independent of modifying reagent are
indicated with ( ). Terminations resulting from base modification or
phosphodiester bond cleavage are one nucleotide shorter than those
resulting from dideoxynucleotide incorporation during the sequencing
reactions. The gels are each representative of four independent sets of
reactions. A, DMS and CMCT modifications. B,
OP-Cu cleavage in the presence of increasing Mg2+
concentrations. C, localization of the modifications and
cleavages.
View larger version (15K):
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Fig. 3.
Nucleotide specificity in the internal
loops. A, the eight bulge loop and hairpin loop
nucleotides of the U-bulge RNA that were randomized for a subsequent
re-selection are indicated in lowercase. B, the sequence of
14 reselected clones and the measured IRP-1 binding affinity relative
to the parental RNA are indicated. Values are the mean and S.D. from
two independent measurements and are all within a factor of three of
the parental. Binding affinities were not determined (ND)
for half the reselected RNAs. C, the reselected bulge loop
sequences of clone 11 (lowercase letters) are also present
within an RNA that was selected from the initial 50N pool and has a G
at the bulge position.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank P. Hardwidge, J. Maher, K. Baron, L. Oppegard, and G. Glick for helpful conversations; M. Hentze for the IRP-1 expression plasmid; M. Yarus and I. Majerfeld for the 50N oligodeoxynucleotide; and S. Eddy for the RNA BOB program.
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FOOTNOTES |
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* This work was supported by grants from the Minnesota Medical Foundation and American Heart Association Grant MN 9706286A.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.
Supported in part by National Institutes of Health Training Grant T32GM07994.
§ To whom correspondence should be addressed. Tel.: 612-624-3132; Fax: 612-625-8408; E-mail: gconnell@lenti.med.umn.edu.
Published, JBC Papers in Press, February 7, 2001, DOI 10.1074/jbc.M010295200
2 RNABOB program.
3 H. Meehan, K. Baron, and G. Connell, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: IRP, iron regulatory protein; IRE, iron responsive element; DMS, dimethylsulfate; CMCT, 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluene sulfonate; RT, reverse transcriptase; OP-Cu, 1,10-phenanthroline-copper complex; PCR, polymerase chain reaction; nt, nucleotide(s); EST, expressed sequence tag.
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