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(Received for publication, October 27,
1995; and in revised form, January 17, 1996) From the
Replication of human immunodeficiency virus type 1 (HIV-1)
requires specific interactions of Tat protein with the trans-activation responsive region (TAR) RNA, a stem-loop
structure containing two helical stem regions separated by a
trinucleotide bulge. The Tat protein contains a basic RNA-binding
region (amino acids 49-57) located in the carboxyl-terminal half
of the protein, and peptides containing this basic domain of Tat
protein can bind TAR RNA with high affinities. We synthesized a
31-amino acid Tat fragment (amino acids 42-72) containing the
basic region and part of flanking regulatory core domain that formed a
specific complex with TAR RNA. Upon UV irradiation (254 nm), this Tat
fragment cross-linked covalently with TAR RNA. Sites of cross-links
were determined on both the TAR RNA and Tat protein fragment by RNA and
protein sequencing, respectively. These results revealed that guanosine
26 of TAR RNA was cross-linked with tyrosine 47 of the Tat peptide. Our
results provide the first physical evidence for a direct amino
acid-base contact in Tat-TAR complex. Recently, orientation of the
Tat-(42-72) was determined in our laboratory by
psoralen
The role of RNA-protein interactions is vital for many
regulatory processes, especially in gene regulation where proteins
specifically interact with binding sites found within RNA transcripts.
RNA molecules can fold into extensive structures containing regions of
double-stranded duplex, hairpins, internal loops, bulged bases, and
pseudoknotted structures(1, 2) . Due to the complexity
of RNA structure, the rules governing sequence-specific RNA-protein
recognition are not well understood. Recent structural studies have
demonstrated that RNA-binding proteins interact with RNA in both the
minor and major grooves. For example, two tRNA synthetases (alanine and
glutamine) interact with the acceptor stems of their cognate tRNAs in
the minor grooves(3, 4) . Major groove recognition
takes place between aspartyl-tRNA synthetase and its cognate tRNAs at a
site of local distortion in the RNA helix(5) . Bulge loops or
bulges (unpaired nucleotides on one strand of a duplex) in RNA helices
are potentially important in tertiary folding of RNA and in providing
sites for specific RNA-protein interactions, as illustrated by TFIIIA
of Xenopus(6) and the coat protein of phage
R17(7) . In a recent report, interactions between U1 small
nuclear RNA and the N-terminal domain of the human U1A protein were
mapped by multidimensional heteronuclear NMR studies(8) . These
studies showed that protein-RNA contacts occur at the single-stranded
apical loop of the hairpin and also in the major groove of the helical
stem at neighboring U-G and U-U non-Watson-Crick base
pairs(8) . Crystal structure of the RNA-binding domain of the
U1A spliceosomal protein complexed with an RNA hairpin also revealed
that the loop sequence (AUUGCAC) interacts with the surface of the
four-stranded The promoter
of the human immunodeficiency virus type 1 (HIV-1), located in the U3
region of the viral long terminal repeat, is an inducible promoter that
can be stimulated by the trans-activator protein,
Tat(12) . As in other lentiviruses, Tat protein is essential
for transactivation of viral gene expression (13, 14, 15, 16) . In the absence of
Tat, most of the viral transcripts terminate prematurely, producing
short RNA molecules ranging in size from 60 to 80 nucleotides. Jeang et al.(17) reported that integrated HIV-1 promoters
did not show a high rate of abortive transcription. Nonetheless, HIV-1
proviruses and integrated long terminal repeats respond efficiently to
Tat(17) . The Tat protein is a small, cysteine-rich nuclear
protein containing 86 amino acids and comprised of three important
functional domains. HIV-1 Tat protein acts by binding to the TAR (trans-activation responsive) RNA element, a 59-base stem-loop
structure located at the 5`-ends of all nascent HIV-1
transcripts(18, 19, 20, 21, 22) .
Upon binding to the TAR RNA sequence, Tat causes a substantial increase
in transcript levels (23, 24, 25, 26, 27) . The
increased efficiency in transcription may result from preventing
premature termination of the transcriptional elongation complex (28) or from enhancing initiation of
transcription(29) . TAR RNA was originally localized to
nucleotides +1 to +80 within the viral long terminal
repeat(18) . Subsequent deletion studies have established that
the region from +19 to +42 incorporates the minimal domain
that is both necessary and sufficient for Tat responsiveness in
vivo(21, 30, 31) . As shown in Fig. 5, the TAR RNA contains a six-nucleotide loop and a
three-nucleotide pyrimidine bulge, which separates two helical stem
regions(18, 21, 22, 25) . The
trinucleotide bulge is essential for high affinity and specific binding
of the Tat protein(32, 33) .
Figure 5:
Mapping
of cross-linked base in the RNA-protein cross-links by alkaline
hydrolysis. A, analysis of 5`-end-labeled TAR RNA and
cross-link: B. cereus ladder of TAR RNA (lane 1);
hydrolysis ladder of TAR RNA (lane 2); hydrolysis ladder of
cross-linked RNA-peptide complex (lane 3). The sequence of TAR
RNA from C
The Tat protein
contains a basic RNA-binding region (amino acids 49-57) located
in the carboxyl-terminal half of the
protein(19, 34, 35, 36, 37) .
Peptides containing the basic domain (residues 49-57) of Tat
protein can bind TAR RNA with high
affinities(36, 38, 39, 40, 41, 42, 43, 44) .
We used a 31-amino acid Tat fragment (amino acids 42-72) to form
a specific complex with TAR RNA. Upon UV irradiation, this Tat fragment
formed a covalent cross-link with TAR RNA. Sites of cross-links were
determined on both the TAR RNA and Tat protein fragment by RNA and
protein sequencing, respectively, which revealed that Tyr
Figure 1:
Separation of covalently cross-linked
Tat-(42-72)
Figure 2:
Effect of Tat peptide concentration on
UV-induced RNA-peptide cross-link formation.
The
photocross-linking reaction between the Tat peptide and TAR RNA was
also dependent on time of irradiation. The yields of cross-linked
RNA-peptide complex were increased with an increase in time of
irradiation (Fig. 3). In this experiment, similar to that shown
in Fig. 2, extended time of irradiation also resulted in the
formation of XL2 at 30 and 40 min. This second minor photoproduct could
be the result of nonspecific binding of the peptide to RNA (at higher
concentrations of peptide) or nonspecific association of photodamaged
RNA and peptide after longer irradiation times. Further
characterization of this minor photoproduct was not carried out in this
study.
Figure 3:
Time course of cross-linking reaction of
Tat-(42-72)
Figure 4:
Specificity of the cross-linking reaction
determined by competition assays. Complexes were formed between 0.25
µM
Figure 6:
Identification of the amino acid in the
Tat-(42-72) sequence that cross-links to TAR RNA. A,
amino acid sequence of the cross-linked peptide. A nonstandard amino
acid (X) was identified during the 6th cycle of N-terminal
sequencing. X in the sequence indicates the cross-linking site
that corresponds to Y in B. B, amino acid
sequence of Tat-(42-72) peptide used in the cross-linking
experiments with TAR RNA. C, schematic representation of the
functional domains of HIV-1 Tat protein. Various numbers refer
to the amino acid positions, and the arrow represents the
separation of the region contributed by adjacent
exon(62) .
Ultraviolet-induced cross-linking of RNA to proteins is a
widely used technique to study in vitro and in vivo RNA-protein interactions(49, 50, 51) .
UV irradiation with sufficient intensity generates a highly reactive
species of RNA, which reacts with protein and organic molecules
involved in making direct contacts with RNA(52, 53) .
To identify specific RNA-protein contacts, we irradiated TAR RNA and
Tat-(42-72) protein complex with UV light and observed the
formation of a covalent bond between RNA and protein. Formation of this
covalently cross-linked product was dependent on the concentration of
Tat peptide and irradiation time ( Fig. 2and Fig. 3). Our
competition and control experiments showed that a specific RNA-protein
complex formation between TAR RNA and Tat fragment was necessary for
photo-crosslinking reactions (Fig. 4). To locate the
cross-link sites in TAR RNA and the Tat peptide, we prepared the
RNA-protein cross-link on a preparative scale, purified the cross-link,
and analyzed it by RNA and protein sequencing. Alkaline hydrolysis of
5`-end-labeled cross-links indicated that a single nucleotide,
G Peptide sequencing on a
tryptic fragment of the cross-link complex was accomplished by Edman
degradation chemistry. The sequencing data indicate that cross-linking
occurred at Tyr It has
been shown by a number of groups that Tat-derived peptides that contain
the basic arginine-rich region of Tat are able to form in vitro complexes with TAR
RNA(36, 38, 39, 40, 41, 42, 43, 44) .
Recently, Churcher et al.(44) published a detailed
comparative study arguing that Tat peptides can mimic the binding
affinity and specificity of Tat protein. Results from that study showed
that the addition of amino acid residues from the core region of the
Tat protein to the arginine-rich domain-containing peptides increased
binding specificities(44) . To achieve specific RNA binding by
a Tat fragment, we used a Tat peptide, Tat-(42-72), that
contained an RNA-binding domain and six amino acids from the core
domain of the Tat protein. In this report, our cross-linking results
have established that this Tat-(42-72) peptide forms a specific
covalent photocross-link to TAR RNA where Tyr What is the biological
relevance of these findings? A number of studies showed that the
immediate stem nucleotide base pairs flanking the bulge region of TAR
RNA are required for Tat binding and trans-activation(44, 55, 56) .
During a detailed mutational analysis of TAR RNA, it was reported that
a change of the G
Figure 7:
A schematic illustration to show a
three-dimensional model of the HIV-1 Tat binding site of TAR RNA and
the location of the amino terminus of Tat-(42-72) and
interactions of Tyr
How does Tat interact with TAR RNA?
Several lines of evidence suggest that Tat protein contacts TAR RNA in
a widened major groove. In a recent study from our laboratory, we used
a rhodium complex, bis(phenanthroline)(phenanthrenequinone
diimine)-rhodium(III) (Rh(phen) To determine the relative orientation of the nucleic acid and
protein in the Tat-TAR complex, we have devised a new method based on
psoralen photochemistry(48) . We synthesized a 30-amino acid
fragment containing the arginine-rich RNA-binding domain of Tat
(residues 42-72) and chemically attached a psoralen at the amino
terminus. Upon near ultraviolet irradiation (360 nm), this synthetic
psoralen peptide cross-linked to a single site in the TAR RNA sequence.
The RNA-protein complex was purified, and the cross-link site on TAR
RNA was determined by chemical and primer extension analyses. Our
results show that the amino terminus of Tat-(42-72) contacts, or
is close to, uridine 42 in the lower stem of TAR RNA(48) . On the basis of the above studies, we suggest a model in which Tat
binds to TAR RNA by inserting the basic recognition sequence into the
enlarged major groove with an orientation where lysine 41 in the core
domain of Tat contacts the lower stem and Tyr
Volume 271,
Number 17,
Issue of April 26, 1996 pp. 10391-10396
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Tat-(42-72) conjugate (Wang, Z., and Rana, T.
M.(1995) J. Am. Chem. Soc. 117, 5438-5444). On the basis
of our findings, we suggest a model in which Tat binds to TAR RNA by
inserting the basic recognition sequence into the major groove with an
orientation where lysine 41 in the core domain of Tat contacts the
lower stem and Tyr
is close to G
of TAR RNA.
The knowledge of the orientation of Tat and details of other
interactions with TAR RNA in Tat-TAR complex has significant
implications for understanding gene regulation in HIV-1.
-sheet (9) . On the basis of NMR data, it
has been shown that TAR (
)RNA in HIV-1 changes its
conformation upon arginine binding(10, 11) . All of
these studies suggest that the diversity of RNA structures plays a
central role in their specific recognition by proteins.
to U
is labeled. A gap in the
sequence is obvious after the U residue, indicating that
G is the cross-linked base. B, sequence and
secondary structure of wild-type TAR RNA used in this study. TAR RNA
spans the minimal sequences that are required for Tat responsiveness in vivo(21) and for in vitro binding of
Tat-derived peptides(38) . Wild-type TAR contains two
non-wild-type base pairs to increase transcription by T7 RNA
polymerase. U25 represents the nucleotide at which the
hydrolysis of the 5`-end-labeled cross-linked RNA-peptide complex was
stopped. The arrow indicates the location of guanosine 26,
which is the cross-linked base in TAR RNA (shown in boldface).
of Tat is close to G of TAR RNA. Our results provide
the first physical evidence for a direct amino acid-base contact in
Tat-TAR complex.
Buffers
All buffer pH values refer to measurements at room
temperature. Transcription buffer contained 40 mM tris-HCl (pH
8.1), 1 mM spermidine, 0.01% Triton X-100, 5 mM dithiothreitol. TBE buffer contained 50 mM Tris, 50
mM boric acid (pH 8.3), 1 mM EDTA. Sample loading
buffer contained 9 M urea, 50 mM Tris, 50 mM boric acid (pH 8.3), 1 mM EDTA, 0.025% bromphenol blue,
0.025% xylene cyanol.Oligonucleotide Synthesis
DNAs
All DNAs were synthesized on an Applied
Biosystems ABI 392 DNA/RNA synthesizer. The template strand encodes the
sequence for the TAR RNA wild type. The top strand is a short piece of
DNA complementary to the 3`-end of all template DNAs having the
sequence 5`-TAATACGACTCACTATAG-3`. DNA was deprotected in
NH
OH at 55 °C for 8 h and then dried in a Savant
Speedvac. The samples were resuspended in sample loading buffer and
were purified on 8 M urea-20% acrylamide denaturing gels, 450
0.8 mm. Gels were run for 4 h at 30 W until bromphenol blue
tracking dye was 5 cm from the bottom of the gel. DNAs were visualized
by UV shadowing, excised from the gel, and eluted in 50 mM Tris, 50 mM boric acid, 1 mM EDTA, and 0.5 M sodium acetate. DNAs were ethanol-precipitated and resuspended in
diethyl pyrocarbonate-treated water. Concentration of DNAs was
determined by measuring absorbance at 260 nm in a Shimadzu UV
spectrophotometer. Samples were stored at -20 °C.RNAs
RNAs were prepared in vitro by
transcription from synthetic DNA templates by T7
polymerase(45) . The template strand of DNA was annealed to an
equimolar amount of top strand DNA, and transcriptions were carried out
in transcription buffer and 4.0 mM NTPs at 37 °C for
2-4 h. For reactions containing 8.0 pmol of template DNA,
40-60 units of T7 polymerase was used. Reactions were stopped by
adding an equal volume of sample loading buffer. RNAs were purified by
electrophoresis on an 8 M urea, 20% polyacrylamide gel as
described above. The sequence of RNAs was determined by base hydrolysis
and nuclease digestion. RNA, starting with 300 pmol, was 5`-end-labeled
with 
P to a specific activity of 2000 10 cpm/pmol after dephosphorylation with calf intestinal alkaline
phosphatase (Promega), as described(46) . The labeled RNAs were
purified by electrophoresis on an 8 M urea, 20% polyacrylamide
gel or by a Sep-Pak C
cartridge (Waters, Millipore Corp.). Peptide Synthesis
All Fmoc (N-(9-fluorenyl)methoxycarbonyl) amino
acids, piperidine, 4-dimethylaminopyridine, dichloromethane, N,N-dimethylforamide, 1-hydroxybenzotriazole,
2-(1H-benzotriazo-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate,
diisopropylethylamine, and HMP-linked polystyrene resin were obtained
from the Applied Biosystems Division, Perkin-Elmer. Trifluoroacetic
acid, 1,2-ethanedithiol, phenol, and thioanisole were from Sigma. Two
Tat-derived peptides (from amino acids 42-72 and 48-72)
were synthesized on an Applied Biosystems 431A peptide synthesizer
using standard Fast Moc protocols. Cleavage and deprotection of the
peptide was carried out in 2 ml of Reagent K for 6 h at room
temperature. Reagent K contained 1.75 ml of trifluoroacetic acid, 100
µl of thioanisole, 100 µl of water, and 50 µl of
ethanedithiol(47) . After cleavage from the resin, the peptide
was purified by high pressure liquid chromatography on a Zorbax 300
SB-C
column. The mass of fully deprotected and purified
peptide was confirmed by fast atom bombardment mass spectrometry:
calculated mass for Tat-(42-72)
C
H
N
O
=
3605.5, found 3606.5 (M + H); calculated mass for
Tat-(48-72) C
H
N
O
= 3000.4, found 3001.4 (M + H).UV Irradiation of RNA-Peptide Complex
A typical cross-linking reaction mixture (15 µl)
contained 0.25 µM labeled RNA, 1.9 µM Tat
peptide, 80 µg/ml bovine serum albumin, 25 mM Tris (pH
7.4), and 100 mM NaCl. RNAs in H
O were heated at
75 °C for 2 min and slowly cooled to room temperature. The reaction
mixtures were incubated at 25 °C for 30 min and cooled on ice
before UV irradiation. The UV irradiation was conducted on ice for 10
min (254 nm; 1280 ergs/mm/s) in a Rayonet RPR 100
photochemical reactor (Southern New England Ultraviolet Inc.). After UV
irradiation, 2 µl of 20 mg/ml yeast tRNA (to remove Tat peptide
from non-cross-linked RNA-peptide complex) and 10 µl of sample
loading buffer were added. The mixtures were heated at 75 °C for 1
min before being loaded on 8 M urea, 20% polyacrylamide gel.
After electrophoresis, the gels were directly autoradiographed.
Efficiencies of cross-linking were determined by a PhosphorImager
analysis (Molecular Dynamics).Base Hydrolysis of RNA Cross-linked with Peptide
5`-end-labeled cross-linked RNA-peptide complexes were
prepared as described above. After electrophoresis, the band
corresponding to the cross-linked RNA-peptide complex, identified by
autoradiography, was excised and eluted into the TBE buffer at 4 °C
overnight. The samples were then ethanol-precipitated and dried in a
Savant Speedvac. Approximately 2 pmol of 5`-end-labeled RNA-peptide
complex was partially hydrolyzed in the presence of 50 mM sodium carbonate (pH 9.2) for 20 min at 85 °C. Size was
assigned to the individual band by comparison with the migration of RNA
digestion products produced by RNase T1 and Bacillus cereus (Pharmacia Biotech Inc.).Purification of Cross-linked Complex, Digestion, and
Peptide Sequencing
Preparation of Cross-linked Complex on a Preparative
Scale
The reaction mixture (10 ml) contained 4 µM 5`-end-labeled RNA, 6 µM peptide, 25 mM Tris
(pH 7.4), and 50 mM NaCl. After UV irradiation, the mixtures
were applied to a Mono Q HR 5/5 column (Pharmacia) equilibrated with 40
mM KCl, 25 mM Tris (pH 8.3) and eluted from the
column with a salt gradient (0-8 min, 40-440 mM KCl; 8-16 min, 440-640 mM KCl; and
16-24 min, 640-1040 mM KCl) at a flow rate of 1 ml/min.
RNA-peptide complex was eluted at approximately 480 mM KCl and
free RNA at 550 mM KCl. The fractions containing RNA-peptide
complex were identified by gel electrophoresis (SDS and urea gels) and
autoradiography. The RNA-peptide complex was desalted on a Sep-Pak
C cartridge and lyophilized.Digestion and Peptide Sequencing
The
5`-end-labeled RNA-peptide complex was dissolved in 0.15 ml of
HO, 80 µl of 0.5 M Tris (pH 8.1), and 80
µl of 1% SDS. After heating at 70 °C for 1 min and cooling to
37 °C, 0.62 ml of HO, 0.067 ml of 15 mM CaCl, and then 0.25 µg of sequencing grade
modified trypsin (Promega) were added. The samples were incubated at 37
°C for 13 h. After phenol extraction and ethanol precipitation, the
samples were dissolved in 70 µl of HO and 70 µl of
sample loading buffer and electrophoresed on 8 M urea, 20%
polyacrylamide. The tryptic fragments were identified by
autoradiography, excised from the gel, eluted into 2 ml of 10 mM ammonium carbonate (pH 8.1), washed exhaustively with
HO on Sep-Pak C cartridge, and dried in a
Savant Speedvac. The concentration of cross-linked RNA-peptide complex
was determined based on RNA absorption at 260 nm. Amino acid sequencing
was conducted at Yale University (New Haven, CT) on an Applied
Biosystems sequencer.
Formation of Cross-linked RNA-Peptide
Complex
Before performing the UV-induced cross-linking
experiment, it was necessary to establish the binding affinities of the
Tat fragment (residues 42-72) used in this study. The
dissociation constant (K
) of TAR RNA and the Tat
peptide was estimated to be 0.13 µM(48) . The
concentrations of TAR RNA and the Tat peptide employed for
cross-linking reactions were 0.25 µM and 1.9
µM, respectively. Under these conditions, electrophoretic
mobility shift assays revealed (data not shown) only one slow migrating
RNA-peptide complex, indicating the absence of other nonspecific
RNA-peptide complex formation(44, 48) . TAR RNA
labeled at the 5`-end with P was incubated with the Tat
peptide for 20 min in 25 mM Tris (pH 7.4) and 100 mM NaCl and UV-irradiated with 254-nm light (see ``Experimental
Procedures''). Products of the photoreaction were analyzed by
denaturing 8 M urea-polyacrylamide gel electrophoresis (Fig. 1). Irradiation of the RNA-peptide complex yields a new
band with electrophoretic mobility less than that of TAR RNA (lane
4). Both the peptide and UV irradiation are required for the
formation of a cross-linked RNA-protein complex. This is evidenced by
the fact that no cross-linked products are observed when RNA is
irradiated in the absence of Tat peptide (lane 2) or incubated
with peptide in the dark without UV irradiation (lane 3).
Further control experiments showed that no cross-linking was observed
when RNA and peptide were irradiated separately and then mixed (lane 5). Digestion of the RNA-peptide cross-link with
Proteinase K (5 units for 30 min at 37 °C) resulted in an RNA
species with mobility similar to TAR RNA (lane 6). The
products of irradiation were also analyzed on a denaturing SDS-15%
polyacrylamide gel. Again, a photoproduct with electrophoretic mobility
less than that of TAR RNA was observed that was dependent on the
presence of RNA and peptide (data not shown). The photoproduct yield is
5% as determined by a PhosphorImager analysis. Since the
cross-linked RNA-peptide complex is stable to alkaline pH (9.5), high
temperature (85 °C) and denaturing conditions (8 M urea,
2% SDS) we conclude that a covalent bond is formed between TAR RNA and
the peptide during the cross-linking reaction.
TAR complex from TAR RNA by denaturing 8 M urea-20% acrylamide gel. Lanes 1 and 2, RNA
without (lane 1) and with (lane 2) UV irradiation; lanes 3 and 4, RNA with Tat peptide without (lane
3) and with (lane 4) UV irradiation; lane 5, RNA
and Tat peptide irradiated separately and then combined; lane
6, treatment of UV-irradiated products (lane 4) with
Proteinase K. Reaction mixtures contained 0.25 µM of
5`-P-end-labeled TAR RNA, 1.9 µM Tat-(42-72), and 100 mM NaCl in 25 mM Tris-HCl buffer, pH 7.4. XL, cross-linked RNA-peptide
complex.
Dependence of the Cross-linking Reaction on the
Concentration of Peptide and Time of Irradiation
Formation of
RNA-peptide photocross-link was dependent on the concentration of Tat
peptide. Here, as in Fig. 1, the major cross-link product (XL1)
has slightly lower electrophoretic mobility than TAR RNA. In Fig. 2, the efficiency of XL1 formation was increased as the
peptide concentration was raised from 0.13 µM to 1.25
µM. At a peptide concentration higher than 1.25
µM, a second minor cross-linked product (XL2) with a lower
electrophoretic mobility than that of XL1 was observed.
P-5`-end-labeled TAR RNA (0.25 µM) was
incubated with Tat peptide at concentrations of 0.13, 0.25, 1.25, 2.5,
and 5.0 µM. The concentration ratios of peptide to RNA are
shown in the figure. For details of reaction conditions see
``Experimental Procedures.'' XL1, cross-linked
RNA-peptide complex, major photoproduct; XL2, cross-linked
RNA-peptide complex, minor photoproduct.
TAR complex. The reaction mixture contained 0.25
µM of P-5`-end-labeled TAR RNA and 1.9
µM Tat peptide and was UV irradiated as described under
``Experimental Procedures.'' After the indicated irradiation
times, aliquots were withdrawn and analyzed on 8 M urea-polyacrylamide gel. XL1, cross-linked RNA-peptide
complex, major photoproduct; XL2, cross-linked RNA-peptide
complex, minor photoproduct.
Specificity of the Cross-link
Formation
Specificity of the cross-linking reaction was
established by competition experiments. Cross-linking reactions were
performed in a 15-µl volume containing 0.25 µM of P-5`-end-labeled TAR RNA, 1.9 µM Tat peptide,
80 µg/ml bovine serum albumin, 25 mM Tris-HCl (pH 7.4),
100 mM NaCl, and up to 10 µM unlabeled competitor
RNA. Cross-linked products were separated by 8 M urea-20%
acrylamide gels and quantitated by PhosphorImager analysis. Fig. 4shows that cross-linking was inhibited by the addition of
unlabeled wild-type TAR RNA and not by a mutant TAR RNA lacking the
trinucleotide bulge. Additional control experiments showed that
cross-linking did not occur between a mutant TAR RNA without
trinucleotide bulge and Tat-(42-72) (data not shown). Therefore,
we conclude that formation of a specific RNA-protein complex between
TAR RNA and Tat peptide is necessary for photocross-linking.
P-labeled TAR RNA and 1.9 µM of Tat-(42-72) in the presence of unlabeled wild-type TAR
RNA (A) or bulgeless mutant TAR RNA (B).
Concentrations of the competitor RNA in lanes 2, 3, 4, 5, 6, and 7 were 0, 0.25, 0.5,
2.5, 5, and 10 µM, respectively. Lane 1 was a
control RNA-peptide complex without UV irradiation. C,
quantitative analysis of competition experiments. The fraction of RNA
in RNA-peptide cross-link was determined by PhosphorImager analysis as
described under ``Experimental Procedures.'' 
,
wild-type TAR RNA competitor; 
, bulgeless mutant TAR
RNA.
Guanosine 26 in TAR RNA Cross-links to Tat
Mapping
of the cross-link site on TAR RNA to single nucleotide resolution was
carried out by partial alkaline digestion of gel-purified RNA-protein
cross-link XL1. Fragment sizes are determined by comparison with RNA
oligonucleotides of defined sequence and length generated by digesting
RNA with RNases T and B. Cereus. Base hydrolysis
of RNA and cross-linked RNA-peptide complex generates a ladder of RNA
degradation products. Bands of cross-linked RNA-peptide complexes
migrate more slowly than corresponding free RNA (Fig. 5A, lanes 2 and 3). Base
hydrolysis of the 5`-end-labeled cross-linked complex (Fig. 5A, lane 3) results in an RNA ladder in
which all fragments up to uridine 25 are resolved. There is an obvious
gap in the hydrolysis ladder after U, indicating that the
fragments above uridine 25 from the 5`-end are linked to the Tat
peptide (Fig. 5A, lane 3). A standard base
hydrolysis ladder was observed for 5`-end-labeled TAR RNA showing
sensitivity to base hydrolysis at all positions, including U in the sequence (Fig. 5A, lane 2). Thus,
we conclude that guanosine 26 of TAR RNA is the site at which
cross-linking occurs (Fig. 5B).Cross-linking Occurs at Tyr
To
identify the amino acid(s) of Tat that are involved in specific
cross-linking with TAR RNA, the cross-linked RNA-peptide complex (XL1)
was prepared on a preparative scale (see ``Experimental
Procedures''), purified from noncross-linked TAR RNA by ion
exchange fast protein liquid chromatography, and digested with trypsin.
The tryptic digest products were purified by 8 M urea, 20%
acrylamide denaturing gels and visualized by autoradiography. We
recovered of Tat100 pmol quantities of a tryptic fragment of XL1 and
subjected it to N-terminal sequencing. The amino acid sequencing data
showed that it had a sequence of
Ala-Leu-Gly-Ile-Ser-X-Gly-Arg-Lys-Lys. This sequence
corresponds to the sequence encompassing amino acids 42-51 in
HIV-1 Tat protein (Fig. 6). X at the 6th position
represents a nonstandard amino acid instead of tyrosine 47 of the Tat
peptide. Thus, cross-linking occurs at tyrosine 47 of the Tat peptide.
, in TAR RNA was involved in covalent interaction (Fig. 5, A and B). The absence of bands in the
hydrolysis ladder after U from the 5`-end of RNA indicates
that the RNA fragments after U are covalently linked to
the Tat peptide and migrate more slowly to create a gap in the standard
hydrolysis ladder. Our results clearly demonstrate that cross-linking
occurs at at G in TAR RNA. of the Tat peptide. As shown in Fig. 6, peptide sequencing identified a nonstandard amino acid X at the 6th position of the cross-linked peptide,
Ala-Leu-Gly-Ile-Ser-X-Gly-Arg-Lys-Lys. This sequence
corresponds to the region encompassing amino acids 42-51 in HIV-1
Tat protein (Fig. 6). The nonstandard amino acid most likely
corresponds to a photomodified tyrosine. Sequencing of proteins by
Edman degradation chemistry requires unmodified amino and carbonyl
groups in the backbone of the peptide. Evidence that the Edman
sequencing reaction was able to continue through Tyr indicates that cross-linking does not occur at these
locations(54) . Therefore, we conclude that the aromatic side
chain or C-
atom in the peptide backbone of Tyr is
involved in the covalent cross-link formation with TAR RNA. of the
peptide contacts G of the RNA.-C
base pair to
C-G base pair resulted in only 12% trans-activation by HIV-1 Tat(56) . These reports
strongly support our finding that G is directly involved
in sequence-specific recognition and trans-activation by HIV-1
Tat protein. However, Tat protein mutants where Tyr was
substituted with Ala or His were functional for trans-activation(57, 58) . These data raise
the possibility that Tyr is not essential for RNA
recognition and that the cross-link formation between Tyr and G could be the result of close proximity and
favorable photochemistry. To address this question, we carried out
cross-linking experiments with a Tat fragment lacking Tyr,
Tat-(48-72), which binds TAR RNA with high
affinities(38, 39, 44) . UV irradiation of
TAR RNA complexed with Tat-(48-72) did not yield any specific
RNA-protein cross-link products (data not shown). These results support
our model of Tat-TAR interactions where the basic recognition sequence
of Tat is located in the major groove of TAR RNA, bringing Tyr in close vicinity of G (Fig. 7). The
cross-link formation between G and Tyr is
likely the result of close proximity, favorable orientation, and
photoreactivity of tyrosine.
of the peptide with G of
the RNA. Orientation of the Tat-(42-72) was determined in our
laboratory by psoralenTat-(42-72) conjugate(48) .
The TAR RNA structure is based on NMR data(63) . Ribbon
structure of TAR RNA is shown in five dark lines. The basic
region of Tat-(47-57) is represented as a barrel positioned in the wide major groove, and the N-terminal region
containing Tat-(42-46) is drawn as a line. Tyrosine 47
is shown directly above the G of TAR RNA
(indicated in black) to demonstrate a close proximity between
Tyr and G. As determined by psoralen-Tat
cross-linking experiments, the amino terminus of Tat-(42-72)
contacts, or is close to, uridine 42 in the lower stem of TAR
RNA(48) ; the amino terminus of the peptide is labeled as
NH, and its proximal base, U
of TAR RNA, is
indicated in black. Structures of TAR RNA were visualized
using Insight II software on an IRIS work
station.
phi
), to
probe the effect of bulge bases on the major groove width in TAR
RNA(59) . This metal complex does not bind double helical RNA
or unstructured single-stranded regions of RNA. Instead, sites of
tertiary interaction that are open in the major groove and accessible
to stacking are targeted by the complex through photoactivated
cleavage(60) . The sites targeted by the rhodium complex have
been mapped to single nucleotide resolution on wild-type TAR RNA and on
several mutants of the TAR RNA containing different numbers of mismatch
bases in the bulge region(59) . A strong cleavage at residues
C and U
was observed on the wild-type TAR RNA
and in mutant TAR RNA containing two mismatch bases in the bulge. No
cleavage at C and U was observed in a
bulgeless TAR RNA and in a one-base bulge TAR RNA. Our studies
establish two important factors involved in Tat-TAR recognition. (i)
There is a correlation between major groove opening and Tat binding. At
least a two-base bulge is required for major groove widening and other
conformational changes to facilitate Tat binding. This cannot be
accomplished by a single base bulge. (ii) The Tat fragment
Tat-(42-72) occupies the major groove of TAR RNA and abolishes
access of the rhodium complex. On the basis of chemical modification
and gel mobility studies, a similar model was suggested earlier by
Weeks and Crothers(55) . Last, Hamy et al.(61) carried out site-specific modifications of functional
groups on TAR RNA and showed that Tat forms multiple specific hydrogen
bonds to a series of dispersed sites displayed in the major groove. is close to
G of TAR RNA (Fig. 7). These findings are
intriguing and suggest a possible mechanism of RNA recognition by Tat.
)
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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