From the Department of Biological Sciences, University of Alberta, Edmonton, Alberta T6G 2E9, Canada
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ABSTRACT |
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The FinO protein regulates the transfer potential
of F-like conjugative plasmids through its interaction with FinP
antisense RNA and its target, traJ mRNA. FinO binds to
and protects FinP from degradation and promotes duplex formation
between FinP and traJ mRNA in vitro. The
FinP secondary structure consists of two stem-loop domains separated by
a 4-base spacer and terminated by a 6-base tail. Previous studies
suggested FinO bound to the smooth 14-base pair helix of stem-loop II.
In this investigation, RNA mobility shift analysis was used to study
the interaction between a glutathione S-transferase
(GST)-FinO fusion protein and a series of synthetic FinP and
traJ mRNA variants. Mutations in 16 of the 28 bases in
stem II of FinP that are predicted to disrupt base pairing did not
significantly alter the GST-FinO binding affinity. Removal of the
single-stranded regions on either side of stem-loop II led to a
dramatic decrease in GST-FinO binding to FinP and to the complementary
region of the traJ mRNA leader. While no evidence for
sequence-specific contacts was found, the results suggest that FinO
recognizes the overall shape of the RNA and is influenced by the length
of the single-stranded regions flanking the stem-loop.
RNA-protein interactions are important in the post-transcriptional
regulation of RNA metabolism and expression. An excellent example is
the FinOP fertility inhibition
system, which controls the transfer frequency of F-like conjugative
plasmids. Expression of the plasmid transfer genes is positively
regulated by the TraJ protein, which is required for activation of
transcription from the major transfer operon promoter, pY. Expression
of traJ is negatively regulated by the combined actions of
the finO and finP gene products. FinP is a
plasmid-specific ~79-base antisense RNA molecule, complementary to
part of the 5'-untranslated region (UTR)1 of traJ
mRNA (1-3), which includes the ribosome binding site and first two
codons of traJ. The FinP secondary structure (GenBank accession number U01159) (4) consists of 2 stem-loop (SL) domains,
separated by a 4-base spacer and terminated by a 6-base tail (Fig.
1). The 5'-UTR of traJ
mRNA forms the mirror image of FinP, with the traJ RBS
and start codon being localized to SLIc (complementary to FinP SLI).
The traJ leader also contains an extensive single-stranded
region and a third stem-loop, SLIII, at its 5' end. Binding of FinP to
the traJ UTR is believed to sequester the traJ
RBS, preventing its translation and repressing plasmid transfer.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Secondary structures of FinP antisense RNA
and the traJ mRNA 5'-UTR (Ref. 4).
Nucleotides 1-34 (shaded in black) and 35-79
(white and gray) constitute synthetic SLI and
SLII of FinP, respectively. The sequences in traJ mRNA
that are complementary to FinP are shaded accordingly, and the
traJ RBS and start codon are indicated. traJ
mRNA variants are named according to the number of nucleotides
extending from their 3' ends.
FinP requires a protein cofactor, FinO, to exert its negative effect on traJ expression. FinO is a 21.2-kDa basic protein (5), which has been shown to bind FinP SLII and traJ mRNA in vitro, increasing the rate of duplex formation (6). Binding of FinO to FinP also protects FinP from RNase E-mediated degradation (7, 8), increasing its steady-state concentration to a level that allows sequestration and reduced expression of the traJ mRNA (9). The F plasmid is derepressed for transfer (i.e. it transfers constitutively) due to the insertion of an IS3 element within its finO gene (10, 11). This effect can be overcome by supplying a functional finO gene from a related plasmid such as R6-5 (5) or R100 (12) in trans, resulting in a 100-1000-fold reduction in F plasmid transfer.
The focus of this study is the specificity of the RNA-protein
interaction between FinP antisense RNA and the FinO protein. Eight
alleles of FinP have been described for F-like conjugative plasmids
(Fig. 2), with sequence identity residing
in the stem, spacer and tail regions and variability in the two loops
(2, 13). Two alleles of FinO exist (13), which show very little sequence variation, but are classified on the basis of their levels of
repression of F-like plasmids which is tied to their levels of
expression (5). FinO is not plasmid-specific, which suggests that the
loops are unimportant for RNA-protein recognition. The FinO protein
does not share homology with any of the protein sequence motifs found
in other RNA-binding proteins (14-16), providing no clues to the amino
acid residues involved in RNA recognition.
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This report presents a preliminary characterization of the RNA targets
recognized by FinO. RNA mobility shift analysis was used to determine
the binding affinity of a glutathione S-transferase (GST)-FinO fusion protein for a series of synthetic FinP and
traJ mRNA variants. High affinity binding by GST-FinO
was shown to be dependent on the presence of SLII, flanked on either
side by single-stranded regions. Unexpectedly, mutations that disrupted base pairing in SLII were tolerated, as was the introduction of internal loops. Furthermore, decreasing the length of the 3' tail reduced GST-FinO binding while altering the sequence had no effect. The
same structural features were recognized in traJ mRNA,
suggesting that FinO does not make sequence-specific contacts with the
RNA, but recognizes its overall shape.
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EXPERIMENTAL PROCEDURES |
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Generation of DNA Templates for in Vitro Transcription of RNAs-- Plasmid pLJ5-13 contains a 105-base pair PCR product with the finP gene fused to the T7 promoter sequence in pUC19 (8). During the construction of pLJ5-13, seven spontaneous mutant clones were obtained with single base mutations in stem II (G42:A, G45:A, C46:T, A47:G, G48:A, G49:A, A51:G) and one clone with a double mutation in stem II (C41:T C46:T). RNAs were generated by in vitro transcription from these BamHI-linearized pLJ5-13 mutant plasmids, resulting in the addition of 7 bases (GGGGAUC) to their 3' ends derived from the BamHI site in the vector. All other RNAs were transcribed from gel-purified PCR products prepared with VentTM DNA polymerase (New England Biolabs) using the appropriate primers and DNA templates, as detailed in Tables I and II. pOX38-Km has been described previously (17).
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Preparation of RNAs--
Uniformly labeled RNAs were prepared by
in vitro transcription of DNA templates in reaction mixtures
containing 1× T7 RNA polymerase buffer (Boehringer Mannheim), 1 unit/µl RNAguard (Amersham Pharmacia Biotech), 10 µCi of
[-32P]UTP (Mandel Scientific), 0.5 mM GTP,
ATP, and CTP, and 0.02 mM UTP at 37 °C for 1 h. DNA
was removed with 10 units of RNase-free DNase I (Boehringer Mannheim)
for 15 min at 37 °C, and the labeled RNA was gel-purified as
described (8). RNA was stored at
20 °C for 1 week without
noticeable degradation. A step of RNA denaturation-renaturation did not
improve or modify the binding affinity of the protein; therefore,
gel-purified RNA was used directly for gel-shift analysis.
Gel-shift Analysis of RNA-Protein Interactions--
pGEX-FO2 (6)
was used for expression of a protein fusion between GST and R6-5 FinO.
GST-FinO was purified using glutathione-agarose affinity as described
(6), except that the cells were lysed using a French press, rather than
by sonication (18). The ability of GST-FinO to bind each RNA was
assessed by RNA mobility shift analysis (6). 7.5 fmol of
32P-labeled RNA was incubated with increasing amounts of
GST-FinO (0-31.9 pmol) in a total volume of 30 µl for 30 min at room
temperature in buffer containing 50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 10 mM NaCl, 100 µg/ml bovine serum
albumin (RNase/DNase-free; Boehringer Mannheim), 7.6 units of RNAguard
(Amersham Pharmacia Biotech), 10% glycerol. Samples were loaded onto a
continuously running (150 V) 6% or 8% nondenaturing polyacrylamide
gel in 1× TBE buffer and electrophoresed at room temperature for
1.5 h. Bands were visualized by autoradiography and
phosphorimaging and quantified using a Molecular Dynamics
PhosphorImager 445 SI. The equilibrium association constant
(Ka) for GST-FinO binding was calculated from the
protein concentration that caused 50% of the labeled RNA to shift in
the gel (6, 19). For RNA variants that gave more than one band shift,
Ka values were calculated by considering all
bound RNA as a single species. Except where noted,
Ka values were calculated from three independent
determinations. The percent binding relative to FinP was calculated as
100 × (Ka variant/Ka FinP).
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RESULTS |
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We have previously shown that a fusion of FinO from the related plasmid, R6-5, to GST is active in vivo and in vitro (6, 18). Furthermore, GST-FinO and FinO obtained from thrombin cleavage of the fusion protein bind to FinP RNA with equal affinity.2 In the present study, the relative equilibrium association constants for GST-FinO binding to FinP variants were determined by performing gel-shift assays in which radiolabeled RNA was combined with increasing amounts of purified GST-FinO protein. Labeled RNA was synthesized by in vitro run-off transcription using PCR-generated templates or cloned fragments (see "Experimental Procedures"). Transcripts prepared from cloned fragments contained 7 additional bases (GGGGAUC) at their 3' ends derived from the BamHI site in the vector used to linearize the plasmids. To ensure that the 7 bases from the vector did not alter the binding constants, FinP RNA and the quadruple mutant, FinP C41:U/C46:U/G66:U/G71:U, with or without the additional 3' bases, were synthesized. The presence of the 7 extra bases at the 3' end had no effect on GST-FinO binding (data not shown).
A comparison of GST-FinO binding affinities to FinP, SLI (nucleotides
1-34, Fig. 1) and SLII (nucleotides 35-79), which were transcribed
from PCR-generated templates, is shown in Fig.
3. The Ka for GST-FinO
binding to FinP was 2.0 × 107
M1, 50-fold higher than that for SLI and
2-fold higher than SLII (summarized in Table
III). These values are higher than
previously reported (6) and likely reflect an increase in the fraction of active GST-FinO in preparations using the French press method for
cell lysis, rather than sonication. Since the fraction of GST-FinO that
is active was not determined, these values may still be underestimated.
As a negative control, incubation of either FinP, SLI or SLII with GST
alone did not result in the formation of shifted species in the gel
(data not shown). This indicated that the complexes formed in the
presence of the fusion protein were the result of RNA interaction with
FinO.
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In agreement with the results of van Biesen and Frost (6), these results suggest that the major determinants for FinO binding reside in SLII, which prompted us to look for differences between SLI and SLII. The most notable structural difference between SLI and SLII is an A-A mismatch within stem I (Fig. 1). SLI A12:U, which creates an A:U base pair (bp) at this site, results in a fully duplexed SLI. Its ability to bind GST-FinO was tested and when compared with SLI with the natural A-A mismatch, was not increased (Table III). Thus, this 11-bp helix was not sufficient for recognition. A second obvious difference between stem-loops I and II is the sequence of the loops. To determine if the loops contributed to the specificity of GST-FinO binding, FinP RNAs from ColB2 and R100-1 were synthesized. ColB2 loop I is identical to F, but differs at 6 of the 7 bases comprising loop II (Fig. 2). R100-1 differs from F at 1 base in loop I and 2 bases in loop II, which is also 1 base smaller than F loop II. The Ka for GST-FinO binding to ColB2 was almost identical to F and was slightly increased for R100-1 (Table III). These results indicate that the sequences of the loops are unimportant for FinO binding.
The sequences of the two FinP stems, although highly conserved among the finP alleles (Fig. 2), differ significantly from each other and could account for the preference of GST-FinO for SLII. The effect of mutations in stem II of full-length FinP on GST-FinO binding were examined, and the binding data are summarized in Table III. Spontaneous single base mutations on the 5' side of stem II (G42:A, G45:A, C46:U) reduced the corresponding affinity constants by no more than 30% when compared with wild-type. In the 5' upper half of stem II, A47:G (naturally present in R100-1) and G49:A did not significantly reduce GST-FinO binding, whereas G48:A and A51:G reduced binding by about 50%. Each of these mutations is expected to disrupt Watson-Crick base pairing, reducing the helical length of stem II. The C41:U/C46:U double mutant, which could lead to formation of two non-Watson-Crick G:U base pairs and maintain the helicity of SLII, showed a 30% reduction in binding constant. To disrupt these potential interactions, FinP variants G66:U/G71:U, C46:U/G66:U/C71:U and C41:U/C46:U/G66:U/G71:U were constructed (Table III). None of these mutations significantly altered GST-FinO binding, suggesting that a continuous duplex is not important for efficient binding. In agreement with this, mutation of 4 consecutive base pairs at the base of stem II (C70:A/G71:U/G72:U/C73:A) and 3 base pairs in the 3' upper half of stem II (C62:A/C63:A/C64:A) had only minimal effects on GST-FinO binding (Table III).
GST-FinO Binding Is Enhanced by Single-stranded Regions on Either
Side of SLII--
Since the stem II point mutants examined did not
significantly alter the binding affinity of GST-FinO, we turned our
attention to the conserved single-stranded regions of FinP: the 5'
leader, spacer, and 3' tail. Removal of 3 bases from the 5' leader
following the initiating G required for T7 RNA polymerase transcription (FinP A2,U3,A4) had little effect on binding affinity (Table IV); however, the spacer and tail regions
proved to be important, as shown in Fig.
4 and summarized in Table IV. Deletion of
either the spacer or 3' tail from SLII had minor to moderate effects on
GST-FinO binding, reducing the Ka values by 1.3-fold and 5.5-fold, respectively. Deletion of both the spacer and 3' tail
from SLII had a profound effect, reducing the binding constant 14-fold.
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In order to determine whether FinO had a nonspecific affinity for single-stranded RNA, the ability of polyuridylic acid to compete with FinP for GST-FinO binding was tested. The results (data not shown) indicate that the GST-FinO/FinP interaction was unaffected by the presence of a large molar excess of polyuridylic acid. These results suggest that the single-stranded spacer and 3' tail make important contributions to a higher order structure recognized by FinO.
The nucleotide sequences of synthetic stem-loops I and II were
arbitrarily chosen (6) such that SLI includes the 5' leader and
stem-loop I, whereas SLII includes the spacer, stem-loop II and 3' tail
(Fig. 1). Since GST-FinO binding to SLII was strongly dependent on the
presence of both single-stranded flanking sequences, the observed low
affinity of GST-FinO for SLI might be due to the absence of a 3'
single-stranded region. To test this hypothesis, SLI variants were
constructed that had either the spacer or tail sequence added to their
3' ends. When assayed for GST-FinO binding, the Ka
values for SLI-spacer and SLI-tail were increased 3-fold and 10-fold,
respectively (Fig. 5; Table IV). These
results clearly demonstrate the importance of the 3' flanking sequence to the structure recognized by GST-FinO.
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The Length, but Not Sequence, of the FinP 3' Tail Is Important for
GST-FinO Binding--
The effect of decreasing the length of the 3'
tail following SLII on GST-FinO binding was examined since addition of
a 7-base extension had no effect (see above). Shortening of the SLII 3' tail from 6 bases (GAUUUU) to 4 bases (GAUU) decreased the
Ka 3-fold to 0.38 × 107
M1 (Fig. 6;
Table IV). A further reduction in tail length to 2 bases (GA) decreased
the Ka another 6-fold to 0.06 × 107 M
1. These results suggest
that a minimum 6-base 3' tail is necessary for efficient binding by
GST-FinO. To determine whether the presence of the 3' tail reflected a
sequence-specific or general requirement for additional bases flanking
SLII, variants SLII-GAAAAA, spacer-SLII-GACA, and FinP G74:C/A75:C were
constructed (Table IV). The sequence of each variant was chosen to
avoid introducing any other obvious secondary structural features.
Comparison of the variant pairs (SLII-GAAAAA with SLII-tail;
spacer-SLII-GACA with spacer-SLII-GAUU and FinP G74:C/A75:C with
wild-type FinP) in Table IV shows that these base transversions in the
3' tail had minor effects on GST-FinO binding. These results indicate
that the length, but not sequence, of the FinP 3' tail is important for
high affinity binding by GST-FinO.
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GST-FinO Recognizes the Same Structural Features in traJ mRNA-- The results of an earlier study (6) showed that GST-FinO binds to a truncated 184-base version of the sense mRNA, traJ, with a Ka similar to that for FinP. The sequence and secondary structure of the first 117 bases of F traJ are shown in Fig. 1. The secondary structure of traJ RNA between nucleotides 33 and 111 is almost identical to FinP, with the following exceptions: SLIc of traJ mRNA has an additional mismatch (A-C), which is paired in FinP SLI; SLIIc of traJ is 2 bases shorter than SLII of FinP, resulting in a 6-base traJ spacer, as compared with 4 bases for FinP.
To further characterize the interaction between GST-FinO and F
plasmid-encoded traJ184 (previously called TraJ211, Ref. 6), a series of 3' truncated traJ variants were created and
their binding to GST-FinO was compared with that of FinP. As seen in Fig. 7, addition of increasing amounts of
GST-FinO led to the conversion of low molecular weight bands to one or
more higher molecular weight bands (see "Discussion" for further
comments). GST-FinO bound traJ184 almost as well as FinP
(95%), with a Ka of 1.9 × 107
M1. Deletion of 74 bases from the 3' end of
traJ184, creating traJ110 (see Fig. 1), yielded a
modest 25% reduction in GST-FinO binding to 1.4 × 107 M
1. Removal of SLIc from
traJ110, which results in a transcribed product of 77 bases
(traJ77; similar to SLII of FinP, Fig. 1) reduced GST-FinO
binding by 48% to 0.73 × 107
M
1. This is similar to the 45% reduction in
the binding constant observed for the removal of SLI from FinP (see
Fig. 3; Table III). Further mutation of traJ77 to eliminate
the spacer residues 3' to SLIIc (A72 to C77), creating
traJ71, decreased the Ka for GST-FinO
binding another 53% to 0.34 × 107
M
1. As with FinP, this result suggests that
SLIIc and its 3' flanking sequence are important determinants for high
affinity GST-FinO binding.
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DISCUSSION |
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This report describes the structural features of RNA recognized by the FinO protein. RNA-binding proteins have generally been shown to target single-stranded regions caused by loops, bulges, and mismatches or between helical stems, rather than the duplexed regions themselves (14, 20, 21). Duplexed regions are necessary, however, for spacing and presentation of the single-stranded nucleotides in the correct orientation (20). The hairpin loops represent an obvious single-stranded region of FinP available for protein binding. Three lines of evidence suggest that FinO does not make sequence-specific contacts with bases in the FinP loops. First, although the loop sequences vary between finP alleles (2), FinO is exchangeable among F-like plasmids (5, 22). Second, the traJ mRNA loops are complementary in sequence to FinP, and yet GST-FinO bound traJ and FinP with nearly equal affinity (Figs. 3 and 7). Third, GST-FinO bound F, ColB2, and R100-1 FinPs with the same relative affinity, even though the sequences of the loops vary considerably, especially within loop II.
The results reported by van Biesen and Frost (6) and in this study indicate that the sequence between nucleotides 35 and 79 (SLII) of FinP is sufficient and necessary for high affinity GST-FinO binding. Stem II is fully duplexed in FinP and presents a relatively poor sequence-specific target for FinO. Attempts to disrupt the helical nature of SLII by introduction of internal loops and base substitutions were freely tolerated, indicating that a continuous duplex is not necessary for FinO binding. In agreement with this, single mutations at three of the sites tested (C41, C46, G49) and at G50, had no effect on FinP repressor activity from the related conjugative plasmid R1, as measured by conjugation frequency (23). Thus, the mutant FinP RNAs are also fully stabilized in vivo by FinO (9). These results indirectly demonstrate that FinO binding is not affected by these base substitutions in vivo, in accord with the results obtained in this study, in vitro.
Earlier studies (6) showed that GST-FinO could bind to a FinP/traJ184 duplex or a duplex formed between SLI and the complementary sequence of traJ mRNA. However, the present results indicate that the 14-bp duplex, SLII alone, was not sufficient for high affinity binding by GST-FinO. These apparently conflicting results can be reconciled by the finding that single-stranded regions adjacent to the duplexed RNA were necessary for binding by GST-FinO. In this respect, FinO resembles the stem-loop binding protein (SLBP), which binds to the 3' end of histone mRNA in mammalian cells (24). Efficient binding by SLBP requires at least three nucleotides each 3' and 5' of a stem-loop. FinO requires at least 6 nucleotides 3' to SLII and as many as 4 nucleotides 5' to SLII, although the length of the 5' spacer was not examined in this study. Like FinO, SLBP does not have a strict loop size requirement, suggesting that specific contacts do not involve the loop. However, unlike the interaction between FinO and FinP, the sequence of the stem and flanking regions is important for SLBP binding.
Since no evidence was obtained for sequence-specific contacts between GST-FinO and FinP, our data suggest that FinO recognizes the overall shape of the RNA conferred by a stem-loop structure, flanked on either side by single-stranded regions. Congruent with this, GST-FinO recognizes the same structural features in traJ mRNA. The requirement for a 6-nucleotide flanking region 3' to SLIIc is fulfilled by the traJ spacer, which is 2 bases longer than the FinP spacer (Fig. 1). In addition the traJ spacer, which differs from the FinP 3' tail at 4 of the 6 bases, can serve as a functional 3' flanking region, indicating that sequence is unimportant for binding by FinO. Addition of the FinP tail sequence (GAUUUU) to the 3' side of SLI conferred moderate GST-FinO binding, although the binding constant was 2-fold lower than that for SLII with the equivalent 3' tail. These results suggest that the RNA conformation recognized by GST-FinO can be adopted quite efficiently by three different sequences: traJ mRNA with its 6-base single-stranded spacer, SLI with the FinP 3' tail, and SLII with the 3' tail.
The functionally related RNA-binding protein, Rom, has also been shown
to recognize RNA in a structure-dependent, rather than sequence-dependent fashion. Rom binds to an unstable
complex formed between the complementary hairpin loops of RNA I and RNA
II of ColE1 plasmids (25). The results of three independent studies indicate that Rom is capable of binding and stabilizing any complex formed by pairs containing fully complementary loop sequences (26-28).
Furthermore, Rom has been shown to extend the average half-life of
general pulse-labeled Escherichia coli mRNA (29). Like
Rom, FinO might have many targets in the cell, but the role of FinO in
promoting duplex formation can occur only when it is bound to
complementary RNAs. GST-FinO binds pBR322 antisense RNA I with an
affinity constant of 1.4 × 107
M1, 70% of that for
FinP.3 The secondary
structure of pBR322 RNA I consists of a 9-base leader followed by three
stem-loop domains, with a 2-base spacer between stem-loops I and II
(30, 31). Although the region of RNA I bound by FinO has not been
determined, it is conceivable that FinO has a nonspecific affinity for
the third stem-loop, which lacks a 3' tail, but is longer than 14 bp.
Further experiments are needed to define the binding site and establish
the specificity of this interaction.
Interestingly, binding of GST-FinO to traJ184 gave rise to
four shifted bands of different mobility, with the largest complex being retained in the well (Fig. 7). The fastest migrating complex was
converted to more slowly migrating complexes with increasing GST-FinO
concentration. GST-FinO interaction with traJ110 resulted in
three distinct complexes, whereas two complexes were formed with either
traJ77 or traJ71. One possible explanation for
the formation of multiple complexes may be stepwise binding of GST-FinO monomers to multiple sites on its RNA target. The bands retained in the
well likely represent complexes formed due to GST-FinO aggregation. Cooperative binding has been reported for the human immunodeficiency virus Rev-RRE interaction (32, 33, 34, 35) and between
p24 (a 220-amino acid truncated polypeptide of the protein kinase, PKR)
and human immunodeficiency virus dsTAR RNA (36). A closer look at
GST-FinO binding to its antisense RNA targets (Fig. 3) shows that only
one complex was formed between GST-FinO and either SLI or SLII, whereas
two complexes were formed with full-length FinP (more readily
distinguishable by under-exposure of this gel; data not shown).
Although the strongest GST-FinO binding was achieved with SLII, low
affinity binding was observed between GST-FinO and SLI with its
attached 4-base spacer. Thus, binding of GST-FinO to its primary
binding site, SLII, may promote binding to the low affinity site, SLI,
resulting in the more slowly migrating complex observed at high
GST-FinO concentrations. Similarly, the higher molecular weight
complexes observed with the traJ RNA variants may represent
successive GST-FinO binding to stem-loops IIc, Ic, and III. Further
experiments are needed to determine the specificity of FinO binding to
these potential low affinity sites. In addition, since the
physiological FinO concentration is not presently known but is thought
to be low, based on mRNA levels (5), the relevance of the higher
molecular weight complexes, which are formed at high FinO
concentrations, remains to be established.
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FOOTNOTES |
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* This research was supported by Grant MT-11249 from the Medical Research Council of Canada (to L. S. F.) and by the Alberta Heritage Foundation for Medical Research (to L. J. J.) and the Izaak Walton Killam Memorial Foundation (to L. J. J.).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: Dept. of Biological
Sciences, CW405 Biological Sciences Bldg., University of Alberta, Edmonton, Alberta T6G 2E9, Canada. Tel.: 780-492-0458; Fax:
780-492-1903; E-mail: laura.frost{at}ualberta.ca.
2 M. J. Gubbins and L. S. Frost, unpublished data.
3 L. J. Jerome and L. S. Frost, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: UTR, untranslated region; SL, stem-loop; SLBP, SL-binding protein; GST, glutathione S-transferase; bp, base pair(s); PCR, polymerase chain reaction.
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REFERENCES |
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