From the Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107
Received for publication, September 29, 2000
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ABSTRACT |
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Zinc finger proteins with high affinity for human
immunodeficiency virus Rev responsive element stem loop IIB (RRE-IIB)
were previously isolated from a phage display zinc finger library. Zinc
fingers from one of these proteins, RR1, were expressed individually and assayed for RRE-IIB affinity. The C-terminal zinc finger retained much of the binding affinity of the two-finger parent and was disrupted
by mutations predicted to narrow the RRE-IIB major groove and which
disrupt Rev binding. In contrast, the N-terminal zinc finger has a
calculated affinity at least 1000-fold lower. Despite the high affinity
and specificity of RR1 for RRE-IIB, binding affinity for a
234-nucleotide human immunodeficiency virus Rev responsive element
(RRE234) was significantly lower. Therefore, zinc
finger proteins that bind specifically to RRE234 were
constructed using an in vitro selection and recombination
approach. These zinc fingers bound RRE234 with subnanomolar
dissociation constants and bound the isolated RRE-IIB stem loop with an
affinity 2 orders of magnitude lower but similar to the affinity of an
arginine-rich peptide derived from Rev. These data show that single
C2H2 zinc fingers can bind RNA specifically and
suggest that their binding to stem loop IIB is similar to that of Rev
peptide. However, binding to RRE234 is either
different from stem loop IIB binding or the tertiary structure of stem
loop IIB is changed within the Rev responsive element.
C2H2 zinc finger proteins comprise a
diverse family of DNA- and RNA-binding proteins. NMR and
crystallographic data show clearly that zinc fingers bind DNA through
The 234-nucleotide HIV RRE (RRE234) found within the
env gene was chosen as a zinc finger target because of the
potential for designing a protein that competitively inhibits Rev
binding and HIV propagation (17). Rev is an essential HIV
protein that induces the accumulation of unspliced and partially
spliced viral RNA in the cytoplasm of infected cells. Rev activity is
dependent upon binding to stem loop IIB of the RRE and subsequent
multimerization (18-20). Rev multimerization is cooperative and
results in a high apparent binding affinity for RRE (21, 22). Thus, an
effective zinc finger-based inhibitor that functions in the absence of
multimer formation could be based on a mixture of zinc fingers that
bind to multiple sites on the RRE in addition to the primary Rev
binding site in stem loop IIB.
Here we report that RR1, a zinc finger protein previously selected for
binding to RRE-IIB, and a single zinc finger derived from it
bind RRE-IIB with similar RNA sequence and structural requirements as
the arginine-rich Rev peptide (amino acids 34-50). In contrast to the
high affinity binding to stem loop IIB, RR1 has lower affinity for the
full-length RRE234. In light of these results, we
constructed high affinity zinc fingers that bind RRE234 by
phage display and in vitro recombination of Phage Display Selection--
Selections for RNA-binding proteins
were done with an Peptide Purification, Gel Shift Analysis, and KD
Determination--
Zinc finger cDNA was amplified by polymerase
chain reaction and cloned into pET28b(+) for expression of N-terminal
(His)6-tagged fusions. All plasmid constructs were
confirmed by automated DNA sequencing. Zinc fingers were expressed in
E. coli BL21 cells, purified by nickel affinity
chromatography, folded by dialysis, and cleaved from (His)6
tags as described previously (16). Gel shift analysis was performed as
described previously, except for the case of full-length RRE, in which
electrophoresis was done for 5 h at room temperature with 8 mA
constant current. KD was determined by plotting the
fraction of RNA bound against protein concentration and curve fitting
to a variation of the Hill equation using SYSTAT (5).
RNA Synthesis--
Overlapping oligonucleotides corresponding to
RRE IIB RNA with or without the G71A and G46:C74 to C46:G74 alterations
were synthesized, annealed, and filled in with the Klenow fragment of
DNA polymerase I. The filled in product included a T7 promoter and a 5'
EcoRI site and a 3' SmaI site, which were used
for subcloning into pUC19. Before RNA production, the resultant
plasmids were digested with SmaI. The RRE IIB RNA
synthesized from these templates included 5' GGG and 3' CCC extensions
(Fig. 2A). The DNA template for production of
RRE234 RNA was made by polymerase chain reaction amplification of the RRE from the plasmid pDM128 (23) with specific primers. The polymerase chain reaction product, which included a 5' T7
promoter, was purified on a Qiagen polymerase chain reaction purification column and used directly for RNA production. All RNAs were
synthesized by in vitro transcription with T7 RNA polymerase using an Ambion MegaShort Kit according to the manufacturer's instructions.
A Single Zinc Finger Is Sufficient for RRE-IIB
Interaction--
Previously, we reported the selection of zinc finger
peptides with high affinity and specificity for both RRE-IIB and 5 S RNA (16). The library used for these selections consisted of two zinc
fingers, both with limited randomization in RRE-IIB Mutations Affect Single-finger Binding--
The dominant
contribution by the basic zinc finger in RR1 suggested that
RRE-IIB-selected zinc fingers could bind RRE-IIB in a manner similar to
that of the arginine-rich Rev34-50 peptide, which makes
Phage Display Selection of RRE-binding Peptides--
RR1 and other
zinc fingers selected to the stem loop IIB region bind a more extensive
234-nucleotide region of the RRE with at least 100-fold lower affinity
than RRE-IIB (Fig. 3). (The zinc finger protein SFR1 was isolated to
the full-length RRE (below) and is shown in Fig. 3 for comparison.)
Furthermore, in some cases (RR1 and RR31), the zinc fingers failed to
form discrete shifted species, suggesting nonspecific RNA affinity or
lower stability during electrophoresis. To isolate zinc fingers that
bind RRE234 and to determine whether other sites on
full-length RRE other than stem loop IIB are conducive for zinc finger
Five rounds of phage display selection were executed in the presence of
a 1000-fold molar excess of tRNA. The percentage of phage retained on
RRE234 increased over five rounds of selection, whereas the
percentage of phage retained in negative control experiments without
RNA remained below 0.1% (Fig.
4A). To improve the affinity of selected proteins, zinc finger DNA from selection round 4 was recombined between further rounds of selection (26, 27). In addition,
DNA used for recombination was doped with unselected randomized
oligonucleotides used to make the library. This provides additional
diversity by introducing new zinc finger sequences in tandem with
preselected zinc fingers. DNA from round 4 of the initial selection
series was amplified, digested, and recombined and then subcloned into
fd.tet.7000 for production of phage. Between rounds 5 and 6, zinc
finger DNA was recombined along with 10% zinc finger library DNA, and
after round 6, DNA was recombined again. Finally, three rounds of phage
display selection without recombination were done (Fig.
4B).
Clones were randomly selected and sequenced after the initial five
rounds of selection and again after the final round of selection and
recombination (Fig. 5B). With
the exception of positions +8 and +10, the C-terminal zinc fingers of
phage recovered from the final round of selection (SFR series) were
identical. Phage sequenced after the fifth round of the initial
selection were more diverse, with only one phage (FR2) containing a
second zinc finger identical to the majority of shuffled clones. As
observed with RRE-IIB- and 5 S rRNA-selected zinc fingers, the
C-terminal Affinity of RRE-binding Peptides--
Three clones from the final
round of selection and recombination, SFR1, SFR4, and SFR12, were
recombinantly produced, and their affinity for RRE RNA was determined
by gel shift analysis using protein titration (Fig.
6A). All three peptides bound
RRE RNA with a KD of <1 nM (Fig.
6B). Rev binds to RRE RNA with a KD of
1-10 nM. These three RRE RNA-selected proteins had lower
affinity for RRE-IIB RNA (KD of around 250 nM) (Fig. 6C), suggesting that these zinc finger
peptides make significant contacts outside the stem loop IIB region of
RRE RNA.
The zinc finger motif has proven to be an excellent framework for
construction of novel specific RNA-binding proteins (16, 28). We have
shown that a combination of phage display and gene shuffling of a zinc
finger library can be used to construct zinc finger proteins with
specific affinity for RRE-IIB, 5 S rRNA, and RRE234. The
RRE-IIB-selected peptide RR1 requires only the second finger for high
affinity binding, and it is likely that all zinc finger peptides thus
far selected exclusively use the C-terminal zinc finger for RNA binding
because this finger is consistently more conserved and basic than the
N-terminal finger. The dominant role for this zinc finger is surprising
because it is closer to the phage surface during selection, and binding
could be expected to be sterically hindered. There is a 2-3-fold
difference in affinity between RR1 and its C-terminal zinc finger,
suggesting that the N-terminal zinc finger may make a specific contact.
Furthermore, in binding assays with RNA mutated at the core Rev binding
site, the two-finger protein forms a discrete shifted species, whereas the single fingers do not.
Mutations that disrupt the G-G base pair that widens the stem loop IIB
helix inhibit Rev, RR1, and RR1zf2 binding, suggestive of a
similar mode of binding. Presumably all of the RNAs that we have used
for selection contain structural elements that allow the McColl et al. (28) have used the zinc finger framework to
stabilize the Rev peptide in an Blancafort et al. (32) have attempted a systematic analysis
of the potential contacts between base pairs in RNA and zinc finger.
Mutations in the Recent kinetic measurements for the binding of Rev to RRE and a number
of derivatives using surface plasmon resonance suggest affinities 1-2
orders of magnitude higher than previous estimates by gel shift or
filter binding assays (22). The KD for the high
affinity site in RRE-IIB is between 10 and 100 pM. Lower
affinity sites for three additional molecules of Rev are in the
KD range of 40-200 nM. Clearly,
potential inhibitors of Rev function that are not based on dominant
negative Rev mutations (33) and do not multimerize must achieve
affinities for RRE-IIB in the picomolar range or alternatively target
other sites of Rev interaction on the RRE. Isolation of zinc fingers
that bind RRE234 up to 250 times better than stem loop IIB
suggests that zinc fingers can be isolated against regions other than
IIB. Conversely, zinc fingers that bind to RRE-IIB with significantly
higher affinity than RRE234 illustrate that zinc finger
binding is sensitive to RNA tertiary structure.
Zinc finger phage display coupled with structure-based design offers a
unique opportunity to study protein-RNA interaction as well as the
potential for construction of high affinity RNA-binding peptides for
possible therapeutic use. Our demonstration that a single zinc finger
can bind specifically to an RNA opens the way to a comprehensive screen
of zinc finger sequence space that was not possible for two-zinc finger proteins.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-helical contacts in a slightly enlarged DNA major groove (1, 2).
Similarly, current evidence suggests that zinc finger proteins
require specific amino acids in
-helices for high affinity
RNA binding (3-5). For example, TFIIIA,1 a nine-zinc finger
protein, binds 5 S rRNA through four central zinc fingers (4-7)
spanning 50-60 nucleotides (5-9). Mutation of amino acids within the
-helices of this group of zinc fingers significantly reduces RNA
affinity. In vitro selection techniques, particularly phage
display, have been used to exploit the modular nature of zinc finger
binding to DNA to design proteins with affinity for new DNA targets
(10-15). In these cases, amino acids within the
-helices of a known
DNA-binding zinc finger protein are changed to make appropriate base
contacts with the new DNA target. To explore the potential for designed
RNA-binding proteins, we previously reported the isolation of zinc
finger proteins with high affinity for HIV RRE-IIB and 5 S rRNA through
a combination of phage display and gene shuffling (16). These zinc
fingers most likely make critical contacts through amino acids in
-helices because these amino acids were randomized in the displayed
zinc finger library.
-helix
randomized zinc fingers.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-helix randomized two-zinc finger library as
described previously (16). Briefly, the phage library was incubated
with biotinylated target RNA for 1 h, and RNA-phage complexes were
immobilized on streptavidin-coated microtiter plates. Phage were eluted
from the plates after extensive washing and amplified in
Escherichia coli K91 cells for additional rounds of
selection. Each selection included a 1000-fold molar excess of yeast
tRNA over biotinylated target RNA. Between selection rounds, zinc
finger cDNA was amplified, recombined (shuffled), and cloned back
into the phage vector (fd.tet.7000), and electroporated into K91 cells
(>106 plaque-forming units) for phage production.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-helix positions
1
through +10 (for sequence examples, see Fig. 5B). Comparison of zinc finger sequences from selected phage showed that the C-terminal zinc finger was consistently basic, whereas the N-terminal zinc finger
was frequently acidic. For example, the
-helix (
2 to +10, see Fig.
5) of the N-terminal zinc finger of RR1 has a calculated pI of 4.2, whereas the
-helix of the C-terminal zinc finger has a pI of 12.2. We sought to determine the contribution of individual zinc fingers to
RNA affinity because many RNA-binding proteins, including the
-helix
from Rev (REV34-50), have basic RNA binding domains. The
N-terminal (zf1) and C-terminal (zf2) zinc fingers from RR1 were
expressed individually, and their affinity for RRE-IIB RNA was measured
by electrophoretic mobility shift assay. RR1zf1 did not bind to
32P-labeled RRE-IIB RNA at protein concentrations as high
as 1500 nM (Fig.
1A). In contrast,
RR1zf2 bound RRE-IIB RNA with an apparent KD
of 19.7 ± 7.9 nM, which is 2-3-fold lower than the affinity of RR1 (KD, 7.9 nM).
RR1zf2-RRE-IIB complexes were not competed with a 1000-fold
molar excess of tRNA or poly(rA) (data not shown). If RR1 zinc
fingers function independently, the C-terminal finger contributes over
95% of the binding energy. Given this assumption, the calculated
affinity of the N-terminal zinc finger is ~300 mM.
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Fig. 1.
Zinc finger 2 of RR1 contributes most of the
energy for RNA binding. A, electrophoretic mobility
shift analysis of RR1 and individual zinc fingers from RR1 (RR1zf1 and
RR1zf2). Radiolabeled RRE-IIB RNA (0.1 nM) was
incubated with the indicated nanomolar concentration of zinc finger
protein, and RNA-protein complexes were separated by nondenaturing
polyacrylamide gel electrophoresis. F, RNA incubated without
protein. KD is reported in nanomolar units ± S.E. of the mean (S.E.M.). B, determination of equilibrium
dissociation constants. The fraction of RNA bound by zinc finger
peptides over a wide range of protein concentrations was determined by
gel shift and quantified using a Molecular Dynamics PhosphorImager.
KD was calculated by fitting data to a variation of
the Hill equation using SYSTAT. RR1zf2, ; RR1,
.
Error bars, S.E. of the mean of at least three
determinations.
-helix-RNA major groove contacts (24). To test this hypothesis, two
mutants of RRE-IIB RNA, G71A and G46:C74 to C46:G74 (Fig.
2A), which greatly reduce the
affinity of the RNA for Rev34-50, were used as substrates
(25). The G71A mutation disrupts a purine-purine base pair responsible for widening the RNA major groove and thereby excludes the
Rev34-50
-helix. Mutation of G46:C74 to C46:G74 is
predicted not to alter the structure of the RNA but instead to change
the sequence of bases that make critical Rev34-50
contacts. Affinity of the two-finger protein RR1 was reduced at least
100-fold by the RRE-IIB mutations (Fig. 2B). In gel shift
assays with purified RR1zf2, C46:G74 and G71A both failed to
form a discrete shifted species, indicative of either greater
nonspecific binding or significantly decreased stability during
electrophoresis (Fig. 3C).
Based on the amount of unshifted RNA, RR1 zinc finger binding is more
severely reduced by the G71A mutation. Thus, it is probable that
selected zinc fingers require distortion of the RNA helix to allow the zinc finger
-helix access to the rich hydrogen bonding potential within the major groove.
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Fig. 2.
Zinc finger binding is sensitive to mutations
in RRE-IIB RNA. A, RRE-IIB RNA was used for binding
assays with nucleotide mutations indicated by arrows.
B, two-finger protein RR1 binding to RRE-IIB RNA.
C, single-finger RR1zf2 binding to RRE-IIB RNA.
Radiolabeled RRE-IIB RNA or mutated RRE-IIB (0.1 nM) was
incubated with the indicated nanomolar concentration of zinc finger
protein, and RNA-protein complexes were separated by nondenaturing
polyacrylamide gel electrophoresis. F, RNA incubated without
protein.
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Fig. 3.
Zinc finger binding to full-length
RRE234. SFR1 is a zinc finger protein selected to
RRE234. RR38, RR1, and RR31 are zinc finger proteins
selected against RRE-IIB. Radiolabeled RRE-IIB RNA (0.1 nM)
was incubated with the indicated nanomolar concentration of protein,
and RNA-protein complexes were separated by nondenaturing
polyacrylamide gel electrophoresis. F, RNA incubated without
protein.
-helix binding, we conducted phage display selection using
immobilized RRE234 RNA.
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Fig. 4.
Selection of full-length RRE zinc finger
peptides. indicates the fraction of input phage recovered when
incubated with biotinylated RRE234 RNA at each successive
round of selection.
indicates corresponding recovery when incubated
without RNA. Selections were done in the presence of 1000-fold molar
excess over biotinylated RRE234 RNA of tRNA. A,
initial RRE234 phage display selection. B,
selection of RRE234 RNA-binding zinc fingers with
recombination. Manipulation of selected DNA between rounds is indicated
by R for recombination and O for addition of 10%
(w/w) original oligonucleotide library DNA.
-helix of the RRE-selected clones was conserved and basic
(pI = 12.19 for clone SFR8), whereas the
-helix of the first
zinc finger was less well conserved and acidic (pI = 6.26). It is
likely that, as in the case of RR1, the second zinc finger of these
peptides contributes most or all of the energy for RNA binding.
Interestingly, lysine is frequently selected over arginine between the
zinc coordinating histidines.
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Fig. 5.
Compilation of
-helix amino acid sequences of RRE- and
RRE-IIB-binding zinc finger peptides. A, helical wheel
plot superimposing ZF2-Rev and RR1zf2. Histidines that are zinc
ligands are aligned in the left panel. In the right
panel, the RR1zf2 helix has been rotated 50°
counterclockwise. Boxed amino acids are crucial for Rev
binding, and identical amino acids are in bold.
B, amino acids at
-helix positions of zinc finger
proteins selected by phage display for affinity to full-length RRE are
shown. Shading indicates
-helix positions made degenerate
in the library. Amino acids encoded by the library are shown at the
bottom of the figure. Bold type indicates a
conserved amino acid in the second zinc finger of RRE-selected
sequences. Italicized amino acids are the necessary
conserved amino acids for zinc finger structure. Dots
indicate zinc finger regions held constant in the library, namely
-sheet and linker amino acids. The ZF2-Rev sequence taken from
McColl et al. (28) and the sequence of RR1 selected
for affinity to RRE-IIB are shown at the bottom. Boxed
amino acids are crucial for RRE-IIB binding by the Rev
peptide.
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Fig. 6.
RRE234 and RRE-IIB RNA binding of
selected and recombined zinc finger proteins. A,
electrophoretic mobility shift assay. Radiolabeled RRE234
RNA (0.01 nM) was incubated with the indicated nanomolar
concentrations of zinc finger protein, and RNA-protein complexes were
separated by nondenaturing polyacrylamide gel electrophoresis.
F, RNA incubated without protein. KD is
reported in nanomolar units ± S.E.M. B, determination
of equilibrium dissociation constants. KD was
calculated as described in the Fig. 1 legend and is reported in
A. SRF4, ; SRF12,
; SRF1,
. Error bars,
S.E. of the mean for at least three determinations. C,
electrophoretic mobility shift assay. RRE234-selected zinc
finger proteins were used in gel shift analysis with
32P-labeled RRE-IIB as outlined in the Fig. 1 legend.
F, RNA incubated without protein.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-helix to
make sequence-specific contacts. TFIIIA binding to 5 S RNA is
guided principally by RNA structure, with significant contacts at loop
A, where three helices stack, and at loop E (8, 29-31). It has yet to
be determined whether randomization of the
-sheet will allow
isolation of peptides with affinity to RNA without a widened major
groove or further enhance the affinity of peptides preselected for
peptide
-helix RNA binding.
-helical conformation. By
positioning the N-terminal Arg of Rev peptide at
1 with respect to
the
-helix, two nonessential arginines were replaced with histidines
required for zinc coordination. The resulting zinc finger, ZF2-Rev,
binds RRE-IIB with a KD of around 300 ± 40 nM at 4 °C, very close to the KD for
a Rev peptide stabilized by flanking alanines (330 ± 20 nM). ZF2-Rev binding in vivo depends on cysteine at position 5, presumably for zinc finger formation, asparagine at
position 22 for G:A base pair contacts, and a wild-type Rev sequence.
Alignment of
-helices from RR1zf2 and ZF2-Rev using zinc
coordinating histidines as a reference point (Fig. 5B)
reveals that of the amino acid positions critical for Rev binding, only
-helix position +1 (arginine) is identical. However, rotating the
RR1zf2 helix with respect to ZF2 in a helical wheel plot aligns four identical amino acids including asparagine 22, suggesting that the
RR1zf2
-helix and ZF2 could make similar RNA contacts. The
rotation is equivalent to aligning the N terminus of RR1zf2 with
the C-terminal half of ZF2-Rev and may suggest that the helix of RR1
does not penetrate as deeply into the major groove of RRE-IIB. Determination of the structure of RR1zf2 bound to RRE-IIB RNA will reveal the exact nature of the peptide-RNA contacts and provide the foundation to further increase affinity using
structure-based design.
-helix of zinc finger 2 in Zif268 (a three-finger protein) were displayed on bacteriophage and selected for
binding to individual RNA triplets embedded within the center of the
9-base pair Zif268 DNA recognition sequence. Only RNA triplets containing a central mismatch, G:A or C:A, were substrates for zinc
fingers. This underscores our hypothesis that the RNA must be distorted
to accommodate the zinc finger
-helix, as is clearly shown in the
Rev peptide-RRE-IIB structure.
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FOOTNOTES |
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* 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.
Present address: Howard Hughes Medical Institute, University of
Pennsylvania School of Medicine, 415 Curie Blvd., Philadelphia, PA 19104.
§ To whom correspondence should be addressed: Kimmel Cancer Institute, Thomas Jefferson University, 233 S. 10th St., Philadelphia, PA 19107. Tel.: 215-503-4504; Fax: 215-923-0249; E-mail: Martyn.Darby@mail.kimmelcancercenter.org.
Published, JBC Papers in Press, October 30, 2000, DOI 10.1074/jbc.M008927200
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ABBREVIATIONS |
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The abbreviations used are: TFIIIA, transcription factor IIIA; RRE-IIB, Rev responsive element stem loop IIB; HIV, human immunodeficiency virus; RRE, Rev responsive element.
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