Proximity of a Tat Peptide to the HIV-1 TAR RNA Loop Region

Transcriptional regulation in human immunodeficiency virus type 1 (HIV-1) requires specific interactions of Tat protein with the trans-activation resp...
0 downloads 0 Views 130KB Size
Download PDF
512

Bioconjugate Chem. 1999, 10, 512−519

Proximity of a Tat Peptide to the HIV-1 TAR RNA Loop Region Determined by Site-Specific Photo-Cross-Linking Zhuying Wang, Ikramul Huq, and Tariq M. Rana* Department of Pharmacology, Robert Wood Johnson Medical School-UMDNJ, and Molecular Biosciences Graduate Program at Rutgers University, 675 Hoes Lane, Piscataway, New Jersey 08854. Received December 3, 1998; Revised Manuscript Received March 8, 1999

Transcriptional regulation in human immunodeficiency virus type 1 (HIV-1) requires specific interactions of Tat protein with the trans-activation responsive region (TAR) RNA, a 59-base stemloop structure located at the 5′-ends of all HIV-1 mRNAs. We have used a site-specific cross-linking method based on 4-thio-uracil (4-thioU) photochemistry to determine the interactions of a Tat peptide, Tat(38-72), with the loop region of TAR RNA under physiological conditions. A TAR RNA construct with a single 4-thioU residue at positions U31 in the loop sequence was synthesized by chemical methods. Upon UV irradiation, 4-thioU at U31 formed a covalent cross-link with the Tat peptide. We did not observe any RNA-RNA cross-link formation. Competition experiments revealed that a specific RNA-protein complex formation was necessary for the RNA-protein cross-linking reaction. Our results demonstrate that, during RNA-protein recognition, the Tat peptide is located in close proximity to O4 of U31 in the TAR RNA loop sequence.

INTRODUCTION

(HIV-1)1

The human immunodeficiency virus type 1 encodes a transcriptional transactivator protein called Tat, which is expressed early in the viral life cycle and is absolutely required for viral replication and progression of disease (1-3). In the absence of Tat, most of the viral transcripts terminate prematurely producing short RNA molecules ranging in size from 60 to 80 nucleotides. The Tat protein is a small, cysteine-rich nuclear protein containing 86 amino acids and an arginine-rich RNAbinding region (amino acids 49-57) located in the carboxy-terminal half of the molecule (1, 4). 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′-end of all nascent HIV-1 transcripts (5). RNA-protein interaction between TAR and Tat converts RNA polymerase II into its processive form and causes a substantial increase in the production of full-length viral transcripts (6-13). TAR RNA was originally localized to nucleotides +1 to +80 within the viral long terminal repeat (LTR) (14). Subsequent deletion studies have established that the region from +19 to +42 incorporates the minimal domain which is both necessary and sufficient for Tat responsiveness in vivo (15-17). TAR RNA contains a six-nucleotide loop and a three-nucleotide pyrimidine bulge which separates two helical stem regions (5, 14, 15, 18). The trinucleotide bulge is essential for high affinity and specific binding of the Tat protein (19, 20). Tat protein binds TAR RNA in vitro with high affinity. Due to difficulties in Tat purification, small Tat peptides that specifically bind TAR RNA with high affinity have been * To whom correspondence should be addressed. Phone: (732) 235-4082. Fax: (732) 235-3235. E-mail: [email protected]. 1 HIV-1, Human immunodeficiency virus type 1; TAR, transactivation response element; EDTA, ethylenediaminetetraacetic acid; 4-thioU, 4-thiouracil; MALDI-TOF MS, matrix-assisted laser desorption ionization/time-of-flight mass spectrometry.

used in many Tat-TAR studies (21-27). In our TatTAR photo-cross-linking studies, we used ADP-1, a Tat fragment (37-72), which binds TAR RNA with high specificity and includes the basic and part of the core regions of Tat (24). To avoid oxidation problems, we deleted the N-terminal cysteine residue from the ADP-1 sequence (26-28). RNA molecules can fold into extensive structures containing regions of double-stranded duplex, hairpins, internal loops, bulged bases and pseudoknotted structures (29, 30). Due to the complexity of RNA structure, the rules governing sequence-specific RNA-protein recognition are not well understood. RNA-protein interactions are vital for many regulatory processes, especially gene regulation where proteins specifically interact with binding sites found within RNA transcripts. Understanding the principles of Tat-TAR interactions is a crucial step for drug design. Although high-resolution NMR information is limited to the TAR RNA component, a structure of the RNA-protein complex is still missing. Therefore, new methods are needed to determine the topology of RNA-protein complexes under physiological conditions. Recently, we have applied photo-cross-linking and affinity cleaving methods to probe RNA folding and RNA-protein interactions under physiological conditions (26-28, 31-34). Chemical synthesis of long oligoribonucleotides containing modified nucleosides, such as 29-bases TAR RNA, is still not very efficient. To circumvent this problem, two short oligoribonucleotides were synthesized and annealed together to form a duplex RNA that contained the trinucleotide bulge to create a structure similar to the Tat binding site in TAR RNA (35-37). Chemically synthesized duplex TAR RNAs without loop residues have been used to study the role of various functional groups in Tat recognition (35-37). Recently, we and others have performed RNA-protein cross-linking studies on synthetic TAR RNA duplex structures containing Tat-binding bulge sequences (26, 38). Here, we synthe-

10.1021/bc980145j CCC: $18.00 © 1999 American Chemical Society Published on Web 05/17/1999

Proximity of a Tat Peptide to TAR RNA Loop Region

Figure 1. (A) Regions of the HIV-1 Tat protein and sequence of the Tat(38-72) peptide. (B) Secondary structure of TAR RNA used in this study. TAR RNA spans the minimal sequences that are required for Tat responsiveness in vivo (15) and for in vitro binding of Tat-derived peptides (43). Wild-type TAR contains two non-wild-type base pairs to increase transcription by T7 RNA polymerase. Site of 4-thioU incorporation, U31, is highlighted in the TAR RNA. Numbering of nucleotides in the RNA corrsponds to their positions in wild-type TAR RNA. Incorporation of 4-thioU at position 31 was accomplished by chemical synthesis of RNA (31, 34). (C) Structure of 4-thiouridine.

sized a TAR RNA construct (Figure 1) with a single 4-thiouridine (4-thioU) residue at position 31 in the loop sequence. 4-thioU is a very useful photoactivatable probe which has been widely used to study RNA structure (for a review, see ref 39). This is the first example of chemical synthesis of functional TAR RNA containing loopresidues and a photoactivatible nucleoside. Upon UV irradiation, this 4-thioU labeled RNA formed a covalent cross-link with Tat(38-72) peptide. No RNA-RNA crosslink formation was observed. Competition experiments revealed that a specific RNA-protein complex formation was necessary for the RNA-protein cross-linking reaction. Our results indicate that, during RNA-protein recognition, the Tat peptide is located in close proximity to O4 of U31 in the TAR RNA loop sequence. MATERIALS AND METHODS

Buffers. All buffer pH values refer to measurements at room temperature. TK buffer: 50 mM Tris-HCI (pH 7.4), 20 mM KCI, and 0.1% Triton X-100. Transcription buffer: 40 mM Tris-HCI (pH 8.1), 1 mM spermidine, 0.01% Triton X-100, and 5 mM DTT. TBE buffer: 45 mM Tris-borate, pH, 8.0, and 1 mM EDTA. Sample loading buffer: 9 M urea, 1 mM EDTA, and 0.1% bromophenol blue in 1× TBE buffer. Binding buffer: 25 mM Tris-HCI (pH 7.5), 100 mM NaCI, 1 mM MgCI2, and 0.1% Triton X-100. Hydrolysis buffer: 50 mM Na2CO3/NaHCO3, pH 9.2. Elution buffer: 1× TBE and 10% sodium acetate (3M), pH 5.5. RNA Synthesis. RNAs were synthesized by chemical and enzymatic methods. Chemical syntheses were performed on ABI synthesizer model 392 using standard

Bioconjugate Chem., Vol. 10, No. 3, 1999 513

protocols. All the monomers of (2-cyanoethyl)phosphoramidites were obtained from Glen Research (Sterling, VA). Site-specific incorporation of 4-thioU at position 31 in TAR RNA was carried out by chemical synthesis of RNA using triazoloU phosphoramidite as a precursor of 4-thioU (31). Synthesis of RNA, deprotection, characterization, and purification were performed as described earlier (31, 34). Briefly, incorporation of 4-thioU in the TAR RNA sequence was confirmed by enzymatic digestion. Enzymatic digestion was carried out by incubating 0.2 A260 unit of purified oligonucleotide at 37 °C overnight with 6 µL (12 µg) of snake venom phosphodiesterase and 2 µL (2 units) of alkaline phosphatase in a total volume of 78.2 µL of 32 mM Tris, pH 7.5, and 15 mM MgCl2. Nucleosides were recovered by adding 10 µL of 3 M NaOAc, pH 5.2, and 234 µL of 95% ethanol, mixing and chilling to -80 °C for 30 min. After centrifugation at 13 000 rpm for 20 min at 4 °C, the supernatant was taken and dried in Speedvac (Savant) and redissolved in 200 µL of water for HPLC analysis. HPLC analysis of nucleosides was performed as described previously (31, 33). All other RNAs were prepared by in vitro transcription (40). 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 (41). For reactions (20 µL) containing 8.0 pmol of template DNA, 40-60 units of T7 polymerase (Promega) was used. Transcription reactions were stopped by adding an equal volume of sample loading buffer. RNA was purified on 20% acrylamide 8 M-urea denaturing gels (41). RNAs were stored in DEPC water at -20 °C. RNAs were 5′-dephosphorylated by incubation with calf intestinal alkaline phophatase (Promega) for 1 h at 37 °C in 50 mM Tris-Cl, pH 9.0, 1 mM MgCl2, 0.1 mM ZnCl2, and 1 mM spermidine. The RNAs were purified by multiple extractions with Tris-saturated phenol and one extraction with 24:1 chloroform:isoamyl alcohol followed by ethanol precipitation. The RNAs were 5′-end-labeled with 0.5 µM [γ-32P]ATP (6000 Ci/mmol) (ICN) per 100 pmol of RNA by incubating with 16 units of T4 polynucleotide kinase (New England Biolabs) in 70 mM TrisHCl, pH 7.5, 10 mM MgCl2, and 5 mM DTT. RNAs were gel purified on a denaturing gel, visualized by autoradiography, and recovered from gels as described earlier (41). Peptide Synthesis. All Fmoc-amino acids, piperidine, 4-(dimethylamino)pyridine, dichloromethane, N,N-dimethylforamide, 1-hydroxybenzotriazole (HOBT), 2-(1Hbenzotriazo-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), diisopropylethylamine, and HMPlinked polystyrene resin were obtained from Applied Biosystems Division, Perkin-Elmer. Trifluoroacetic acid, 1,2-ethanedithiol, phenol, and thioanisol were from Sigma. Tat-derived peptide (from amino acids 38 to 72) was synthesized on an Applied Biosystems 431A peptide synthesizer using standard FastMoc 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 TFA, 100 µL of thioanisole, 100µL of water, and 50 µL of ethanedithiol (42). After cleavage from the resin, peptide was purified by HPLC on a Zorbax 300 SB-C8 column. The mass of fully deprotected and purified peptides were confirmed by FAB mass spectrometry; calculated mass for Tat(38-72) C175H298N64O43 ) 4082.7, found ) 4083.7 (M + H). Gel Retardation Assays. RNA-protein binding reactions (20 µL) contained 0.05 µM wild-type, modified TAR, or wild-type TAR RNA and 0-1 µM Tat(38-72) in TK

514 Bioconjugate Chem., Vol. 10, No. 3, 1999

Figure 2. Binding of Tat(38-72) peptide to TAR RNA (A) or 4-thioU-31-modified TAR RNA (B). Binding reactions contained 0.05 µM of 5′-32P-end-labeled RNA and increasing concentration of Tat peptide. Lane 1 was a control lane without the peptide. Complex formation was performed in the TK buffer and incubation at room temperature for 1 h. Complexes were separated from unbound RNA by electrophoresis in nondenaturing 8% polyacrylamide gels containing 0.1% Triton X-100. Gels were run in cold room at 300 V for 2 h. The relative amounts of free and bound RNA were determined by phosphor imaging. RNApeptide complexes are shown as R-P.

buffer. After 15 min of incubation at room temperature, the complexes were resolved on a nondenaturing 8% acrylamide gels and quantified as described earlier (26). Site-Specific Photo-Cross-Linking Reactions. Typically, 5 pmol of TAR RNA (5′-end labeled with 32P) was mixed with 6 pmol of Tat (38-72) in TK buffer, incubated at room temperature for 10 min, and UV irradiated (360 nm) for 5 min on ice in a Rayonet Photochemical Reactor (RPR-100). After irradiation, 2 µL of Yeast tRNA (20 µg/ µL) and 12 µL of sample loading buffer were added to each sample and electrophoresed on 8 M urea-20% acrylamide gels. Efficiencies of cross-linking were determined by a Phosphor Image analysis (Molecular Dynamics). RNA Sequencing. Alkaline hydrolysis of RNAs was carried out in hydrolysis buffer for 8-12 min at 85 °C. RNAs were incubated with 0.1 units RNAse from Bacillus cereus (Pharmacia) per pmol of RNA for 4 min at 55 °C in 16 mM sodium citrate, pH 5.0, 0.8 mM EDTA, 0.5 mg/ mL yeast tRNA (Gibco-BRL). This enzyme yields U and C specific cleavage of RNA. Sequencing products were resolved on 20% denaturing gels and visualized by phosphor image analysis. RESULTS AND DISCUSSION

Tat Binding To Modified TAR RNA. To characterize and evaluate the Tat binding capabilities of 4-thioU containing TAR RNA, we determined the dissociation constants of modified TAR-Tat complexes. Equilibrium dissociation constants of the Tat:TAR complexes were measured using direct electrophoretic mobility assays (24, 27, 33). A Tat fragment, Tat(38-72), was used in these measurements. A typical gel of these experiments is shown in Figure 2. These results showed that Tat(3872) binds the 4-thioU-31 modified TAR RNA with a KD of 0.22 ( 0.05 µM. To compare the Tat-binding affinities of the modified RNA to wild-type RNA, we synthesized an unmodified RNA (Figure 1). Dissociation constants of the unmodified TAR:Tat complexes were determined under the same conditions used for 4-thioU31-TAR: Tat(38-72) complexes. These experiments showed that

Wang et al.

Figure 3. Cross-linking reactions of TAR RNA labeled with 4-thioU at position 31. TAR RNA was labeled with 32P at the 5′-end and UV irradiated at 360 nm. Cross-linked products were resolved on a 20% acrylamide-8 M urea gel and visualized by autoradiography. Lane 1: RNA without irradiation. Lane 2: RNA with irradiation. Lane 3: RNA irradiated with 1 mM L-argininamide. Lane 4: RNA with Tat(38-72) and no irradiation. Lane 5: RNA irradiated in the presence of Tat(38-72). Lane 6: RNA irradiated in the presence of Tat(38-72) and the sample was treated with Proteinase K for 15 min at 55 °C. RNA-protein cross-links are indicated as XL.

the unmodified TAR duplex binds Tat with a KD of 0.13 ( 0.08 µM (Figure 2). A relative dissociation constant (Krel) can be determined by measuring the ratios of the modified TAR to wild-type TAR dissociation constants (KD) for Tat(38-72). Our results demonstrate that the calculated value for Krel was 1.7. These results indicate that incorporation of 4-thioU in the TAR loop sequence did not significantly alter the structure of the RNA, thus preserving the Tat-binding affinities of the RNA. TAR RNA Labeled with 4-thioU at U31 Forms Cross-Link with a Tat Fragment. Specific RNAprotein complexes were formed by adding the approximately equimolar amounts of TAR RNA and the Tat peptide. Under these conditions, electrophoretic mobilityshift assays revealed only one slow-migrating RNApeptide complex, indicating the absence of other nonspecific RNA-peptide complex formation (24, 32). TAR RNA labeled at its 5′-end with 32P was incubated with the Tat peptide for 10 min in TK buffer and UV irradiated with 360 nm light (see Materials and Methods). Products of the photoreaction were analyzed by denaturing 8 M urea PAGE (Figure 3). Irradiation of the RNA-peptide complex yields band with electrophoretic mobility less than that of RNA (Figure 3, lane 5). Both the peptide and UV irradiation are required for the formation of this crosslinked RNA-protein complex because this cross-linked product is observed only when RNA is irradiated in the presence of Tat peptide (lane 5), and no cross-link is formed with peptide in dark without UV irradiation (lane 4). The formal possibility that the new mobility is due to an alternative RNA-RNA cross-link was ruled out by perfroming proteinase K digestion of the RNA-protein cross-link. RNA-protein cross-link products are degraded by proteinase K enzyme while RNA-RNA cross-link species are stable under these conditions (26, 28). Proteinase K digestion of the RNA-protein cross-link resulted in a loss of RNA-protein cross-link and a gain in free RNA as observed by band intensities on the gel (Figure 3, 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, which was dependent on the presence of RNA and peptide (data not shown). The photoproduct yields were ∼37% as determined by a Phosphor Image analysis. Since the cross-linked RNApeptide complex is stable to alkaline pH (9.2), high temperature (85 °C), and denaturing conditions (8 M urea, 2% SDS), we conclude that a covalent bond is

Proximity of a Tat Peptide to TAR RNA Loop Region

Bioconjugate Chem., Vol. 10, No. 3, 1999 515

Figure 4. Specificity of the Tat-TAR cross-linking reaction determined by competition assays. RNA-protein complexes were formed between 0.25 µM 32P-5′-end labeled TAR RNA modified with 4-thioU at position 31 and 0.5 µM of Tat(38-72) in the presence of unlabeled wild-type TAR RNA (A) or bulge mutant TAR RNA (B). Concentrations of the competitor RNA in lanes 2, 3, 4, 5, and 6 were 0, 0.5, 0.75, 1.0, and 1.25 µM, respectively. Lane 1 was a control RNA-peptide complex without UV irradiation. RNA-protein cross-link is shown as XL.

formed between TAR RNA and the peptide during the cross-linking reaction. Specificity of the RNA-Protein Cross-Link Formation. Specificity of the cross-linking reaction was established by competition experiments (28). Crosslinking reactions were performed in a 10 µL volume containing 0.25 µM of 5′-32P-labeled 4-thio-U31 modified TAR RNA, 0.5 µM Tat peptide, 25 mM Tris.HCl (pH 7.4), 100 mM NaCl, and up to 1.25 µM unlabeled competitor RNA. Cross-linked products were separated by 8 M urea20% polyacrylamide gels and visualized by phosphor image analysis. Figure 4 shows that cross-linking was inhibited by the addition of unlabeled wild-type TAR RNA and not by a bulge mutant TAR RNA. Therefore, we conclude that formation of a specific RNA-protein complex between 4-thioU modified TAR RNA and Tat is necessary for photo-cross-linking. 4-Thio-Uridine 31 in TAR RNA Cross-Links to Tat(38-72). Mapping of the cross-link site on TAR RNA to single nucleotide resolution was carried out by partial RNAse digestion and alkaline hydrolysis of the gelpurified RNA-protein cross-link. Fragment sizes are determined by comparison with RNA oligonucleotides of defined sequence and length generated by digesting RNA with RNases T1 and B. cereus. Alkaline hydrolysis of RNA and cross-linked RNA-peptide complex generates a ladder of RNA-degradation products. Bands of crosslinked RNA-peptide complexes migrate slower than corresponding free RNA (lane 3 in Figure 5). Base hydrolysis of the 5′-end-labeled cross-linked complex (lane 3 in Figure 5) results in an RNA ladder in which all fragments up to C30 are resolved. There is an obvious gap in the hydrolysis ladder after C30, indicating that the fragments above C30 from the 5′-end are linked to the Tat peptide (Figure 5, lane 3) that is not seen with the un-cross-linked RNA (lane 2). On the basis of these results, we conclude that 4-thio-U31 of TAR RNA is indeed the site in RNA sequence at which cross-linking occurs. The basic regions of tat proteins are directly involved in RNA binding. Short basic peptide mimics of this region bind TAR RNA in the bulge region (21, 23-25, 43). A number of studies determined the sequence requirements within the basic region of Tat for RNA binding. In vivo experiments using full-length Tat and in vitro studies with synthetic peptides indicate that conservation of overall positive charge including several arginines is essential for TAR RNA recognition. For example, TatTAR interactions are not affected by interchanging the basic region sequences of Tat and Rev (44). Amino acid substitutions that recreate the consensus sequence of the

Figure 5. Mapping of cross-linked base in the RNA-protein cross-links by alkaline hydrolysis. Analysis of the RNA-protein cross-link containing 5′-end labeled TAR RNA: B. cereus ladder of TAR RNA (lane 1); hydrolysis ladder of TAR RNA (lane 2); hydrolysis ladder of the RNA-peptide cross-link (lane 3). Sequence of the TAR RNA from U23 to U31 is labeled, and a gap in the sequence is obvious after C30 residue indicating that indeed U31 is the cross-linked base.

RNA-binding region (R/KXXRRXRR, where R is arginine, K is lysine, and X is any amino acid) reconstitute a functional Tat protein. These results are in agreement with in vitro peptide studies showing that high-affinity TAR RNA binding requires three of four arginines within the RRXRR stretch, although any one arginine can be substituted, suggesting some redundancy (25). These studies raised the possibility that several amino acids from the RNA-binding region of Tat could be cross-linked to 4-thioU31 in TAR RNA. Although a discrete band for the RNA-protein cross-link was observed in our experiments (Figures 3-5), it is possible that more than one cross-link product is present in the band. To determine the homogeneity of the cross-link products, we analyzed the products by reversed-phase HPLC. Our chromatography data showed that the RNA-protein cross-link products eluted in a single sharp peak from the HPLC column under various solvent systems and gradients (data not shown). These results indicate that homogeneous RNA-protein cross-link products were formed during photo-cross-linking reactions. Models for Tat-TAR Recognition. Tat protein recognizes the trinucleotide bulge in TAR RNA. Key elements required for TAR recognition by Tat have been defined by extensive mutagenesis, chemical probing, and peptide binding studies (21-24, 36, 37, 43, 45-48). Tat interacts with U23, and two other bulge residues, C24 and U25, act as spacers because they can be replaced by other nucleotides or linkers (24, 35). Previous studies suggest that Tat protein contacts TAR RNA in a widened major groove (21, 26, 41). A rhodium complex, Rh(phen)2phi3+, was used to probe the effect of bulge bases on the major groove width in TAR RNA (41). This study showed two important results for 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) A Tat peptide occupies the major groove of TAR RNA and abolishes

516 Bioconjugate Chem., Vol. 10, No. 3, 1999

Wang et al.

Figure 6. Proposed model for Tat peptide-TAR interactions showing protein orientation and the proximity of U31 to the peptide in the RNA-protein complex. Ribbon structure of TAR RNA is shown in yellow lines and nucleotides in red (52). Ribbon structure of the Tat peptide (ribbon/tube) and the amino terminal Phe38 are shown in cyan color. Ribbon structure of the Tat peptide is drawn from Tat protein structure (53). Orientation of the Tat peptide is based on previous photo-cross-linking results indicating that Lys41 and Arg57 are close to U42 and U31, respectively (28, 32). Lys41 and Arg57 side chains are shown in green. U42 is shown in yellow and U31 in atom-by-type colors. It is evident that O4 (red spacefill sphere) of U31 is close to Arg57 side chain of the Tat peptide. Structures of RNA and protein were visualized using Insight II software on an IRIS workstation.

access of the rhodium complex (41). In addition to the trinucleotide bulge region, two base pairs above and below the bulge also contribute significantly to Tat binding (21, 24). Phosphate contacts below the bulge at positions 22, 23, and 40 are critical for Tat interactions (23, 24, 36). In a chemical cross-linking study, Gait and co-workers showed that a TAR duplex containing a trisubstituted pyrophosphate replacing the phosphate at 38-39 reacted specifically with Lys51 in the basic region of Tat(37-72) peptide (38). We carried out 4-thiouracilmediated photo-cross-linking studies on Tat-TAR complex and showed that Tat interacts with U23, U38, and U40 in the major groove of TAR RNA (26). Our recent

cross-linking experiments using 6-thioG-modified TAR RNA demonstrated that Tat interaction with G21 and G26 also takes place in the major groove of RNA (49). Taken together, these studies establish that Tat binds TAR RNA at the trinucleotide bulge region and interacts with two base pairs above and below the bulge in the major groove of RNA. The orientation of a Tat peptide in the Tat-TAR complex was determined by a new method based on psoralen photochemistry (32). A psoralen was chemically attached at the amino terminus of a Tat(42-72) peptide. Upon near-ultraviolet irradiation (360 nm), this synthetic psoralen-peptide cross-linked to a single site in TAR RNA

Proximity of a Tat Peptide to TAR RNA Loop Region

sequence. The RNA-protein complex was purified, and the cross-link site on TAR RNA was determined by chemical and primer extension analyses. These results showed that the amino terminus of Tat(42-72) contacts, or is close to, uridine 42 in the lower stem of TAR RNA (32). To define the relative orientation of the C-terminal part of the arginine-rich domain of Tat, we synthesized Tat(38-72) and replaced Arg57 with Cys to introduce a unique thiol group (-SH) in the peptide (26). A psoralen derivative which can react with thiol groups was synthesized and used for specific chemical modification of Cys57-Tat(38-72). We used this psoralen-Tat conjugate [psoralen-Cys57-Tat(38-72)] to form a specific complex with TAR RNA. Photo-cross-linking and RNA sequencing results revealed that Cys57 of Tat(38-72) is close to U31 of TAR RNA (26). In a recent report from our laboratory, we applied a site-directed RNA-cleaving strategy to determine the neighborhood of the core domain of a Tat fragment in Tat-TAR complex (27). We attached an EDTA analogue to the amino terminus of Tat(38-72) by standard peptide synthesis methods and used this EDTA-Tat conjugate to form a specific complex with TAR RNA. This sequencespecific RNA-binding peptide was converted into a sequence-specific RNA-cleaving peptide by the addition of Fe(II) salt, ascorbate, and H2O2. Hydroxyl radicals generated from the tethered Fe(II) cleaved TAR RNA backbone in two localized regions. Site-specific cleavage of TAR RNA was observed at the bulge residues (U23, C24, and U25), in the loop region (G34 and A35), and at the strand opposite the bulge (U40 and C41). These results demonstrate that in the three-dimensional structure of TatTAR complex, the Phe38 of Tat(38-72) is located in the proximity of the bulge region and two nucleotides from the loop sequence (27). Here we show that 4-thioU at position 31 cross-links to the Tat peptide. Results from the above studies are summarized in a model for Tat-TAR complex showing the protein orientation, location of N-terminus of the peptide, and U31 functional groups (Figure 6). Our attempts to locate the cross-linked amino acid in the Tat peptide by protein sequencing were not successful. This is not an unexpected result due to the length of the fragment and relatively harsh conditions of peptide sequencing which could result in the destruction of the cross-link between the peptide and RNA (50). Previously, we used 4-thioU modified TAR RNA to form Tat-TAR cross-links and we were unable to locate the cross-linked amino acid by Edman protein sequencing (26). Recently, Farrow et al. (51) applied MALDI-TOF mass spectrometric analysis to locate the cross-link sites in the basic region of a Tat peptide to chemically modified TAR RNA. Farrow et al. (51) used a model duplex TAR RNA, and the RNA-protein cross-linked products contained 1417 nucleotide long RNAs. We followed the methods of Farrow et al. (51), and prepared RNA-protein crosslinked products on a large scale and subjected to the MALDI-TOF MS analysis. Due to the larger size of our RNA-protein cross-linked complex, we were unable to analyze it by MALDI-TOF MS. Since our previous findings showed that a psoralen attached to Arg57 formed a cross-link with U31 (28), Arg57 is displayed as a potential side chain located in proximity to U31 of TAR RNA. It is important to note that, in previous studies, psoralen was attached to cysteine 57 by a linker of ∼10 Å and psoralen could have preferably photoreacted with uridines in RNA (28). Although psoralen photo-crosslinking provides useful information about protein orientation in the Tat-TAR complex, direct interactions and

Bioconjugate Chem., Vol. 10, No. 3, 1999 517

close proximity between the RNA and protein have not been established. Experiments presented in this report show that 4-thioU31 directly contacts Tat peptide. A refined model for Tat-TAR interactions is presented in Figure 6 showing protein orientation and close proximity of U31 to an Arg residue. The RNA structure is derived from ref 52, and the ribbon structure of Tat(38-72) peptide is based on Tat protein structure (53). The Tat peptide is positioned in the major groove of TAR RNA and the RNA is folded to bring O4 of U31 within 5 Å of Arg57 side chain. All other structural constraints are based on previous photo-cross-linking and affinity cleaving experiments (26-28, 32). Due to technical difficulties, we were unable to determine which amino acid formed the cross-link with U31. However, Arg57 is the only amino acid which can be placed in close proximity of U31 in order to generate a structure which is consistent with previous cross-linking and affinity cleaving results. Mutational analyses have shown that sequences in the loop of TAR RNA are required for trans-activation (54, 55) and not for Tat binding (35, 36, 43). Tat binds duplex TAR RNA sequences with approximately half the affinity for wild-type TAR RNA (36). Duplex TAR RNA sequences have been used previously to study RNA folding and RNA-protein interactions (26, 38, 56). Recently, Jones and co-workers have identified a novel CDK9-associated C-type cyclin, cyclin T1, which interacts with Tat and increases its affinity and specificity for TAR RNA (57). Formation of a specific and high-affinity ternary complexes (Tat:TAR:Cyclin T1) requires sequences in the loop of TAR RNA (57). Whether Tat protein or cyclin T1 forms a cross-link with 4-thioU31-modified TAR or not, remains to be determined. ACKNOWLEDGMENT

We wish to thank Drs. Michael Gait and Thomas Barlow for MALDI-TOF MS analysis and helpful discussions. This work was supported in part by the National Institutes of Health Grants AI 41404 and TW00702 and a Research Career Development Awrad to T.M.R. LITERATURE CITED (1) Jones, K. A., and Peterlin, B. M. (1994) Control of RNA Initiation and Elongation at The HIV-1 Promoter. Annu. Rev. Biochem. 63, 717-743. (2) Cullen, B. R. (1998) HIV-1 auxiliary proteins: making connections in a dying cell. Cell 93, 685-692. (3) Emerman, M., and Malim, M. (1998) HIV-1 regulatory/ accessory genes: keys to unraveling vial and host cell biology. Science 280, 1880-1884. (4) Cullen, B. R. (1992) Mechanism of action of regulatory proteins encoded by complex retroviruses. Microbiol. Rev. 56, 375-394. (5) Berkhout, B., Silverman, R. H., and Jeang, K. T. (1989) Tat trans-activates the Human Immunodeficiency Virus Through a Nascent RNA Target. Cell 59, 273-282. (6) Kao, S.-Y., Calman, A. F., Luciw, P. A., and Peterlin, B. M. (1987) Antitermination of transcription within the long terminal reppeat of HIV-1 tat gene product. Nature 330, 489493. (7) Laspia, M. F., Rice, A. P., and Mathews, M. B. (1989) HIV-1 tat rotein increases transcriptional initiation and stabilises elongation. Cell 59, 283-292. (8) Feinberg, M. B., Baltimore, D., and Frankel, A. D. (1991) The role of Tat in the human immunodeficiency virus life cycle indicates a primary effect on transcriptional elongation. Proc. Natl. Acad. Sci. U.S.A. 88, 4045-4049. (9) Kato, H., Sumimoto, H., Pognonec, P., Chen, C.-H., Rosen, C. A., and Roeder, R. G. (1992) HIV-1 Tat Acts as a

518 Bioconjugate Chem., Vol. 10, No. 3, 1999 Processivity Factor in Vitro in Conjuction with Cellular Elongation Factors. Genes Dev. 6, 655-666. (10) Marciniak, R. A., Calnan, B. J., Frankel, D. A., and Sharp, P. A. (1990) HIV-1 Tat Protein Trans-Activates Transcription in Vitro. Cell 63, 791-802. (11) Marciniak, R. A., and Sharp, P. A. (1991) HIV-1 Tat protein promotes formation of more-processive elongation complexes. EMBO J. 10, 4189-4196. (12) Parada, C. A., and Roeder, R. G. (1996) Enhanced processivity of RNA polymerase II triggered by Tat-induced phosphorylation of its carboxy-terminal domain. Nature 384, 375378. (13) Cujec, T. P., Cho, H., Maldonado, E., Meyer, J., Reinberg, D., and Peterlin, B. M. (1997) The human immunodeficiency virus transactivator Tat interacts with the RNA polymerase II holoenzyme. Mol. Cell. Biol. 17, 1817-1823. (14) Rosen, C. A., Sodroski, J. G., and Haseltine, W. A. (1985) Location of Cis-acting Regulatory Sequences in the Human T cell Lymphotropic Virus type III (HTLV-III/LAV) Long Terminal Repeat. Cell 41, 813-823. (15) Jakobovits, A., Smith, D. H., Jakobovits, E. B., and Capon, D. J. (1988) A Discrete Element 3′ of Human Immunodeficiency Virus 1 (HIV-1) and HIV-2 mRNA Initiation Sites Mediates Transcriptional Activation by an HIV Trans Activator. Mol. Cell. Biol. 8, 2555-2561. (16) Selby, M. J., Bain, E. S., Luciw, P. A., and Peterlin, B. M. (1989) Structure, Sequence, and Position of the Stem-loop in Tar Determine Transcriptional Elongation by Tat through the HIV-1 Long Terminal Repeat. Genes Dev. 3, 547-558. (17) Garcia, J. A., Harrich, D., Soultanakis, E., Wu, F., Zmitsuyasu, R., and Gaynor, R. B. (1989) Human Immunodeficiency Virus Type 1 LTR TATA and TAR Region Sequences Required for Transcriptional Regulation. EMBO J. 8, 765-778. (18) Muesing, M. A., Smith, D. H., and Capon, D. A. (1987) Regulation of mRNA by a Human Immunodeficiency Virus trans-activator protein. Cell 48, 691-701. (19) Dingwall, C., Ernberg, I., Gait, M. J., Green, S. M., Heaphy, S., Karn, J., Lowe, A. D., Singh, M., Skinner, M. A., and Valerio, R. (1989) Human Immunodeficiency Virus Tat protein Binds Trans-activation-responsive Region (TAR) RNA in vitro. Proc. Natl. Acad. Sci. 86, 6925-6929. (20) Dingwall, C., Ernberg, I., Gait, M. J., Green, S. M., Heaphy, S., Karn, J., Lowe, A. D., Singh, M., and Skinner, M. A. (1990) HIV-1 Tat Protein Stimulates Transcription by Binding to the Stem of the TAR RNA Structure. EMBO J. 9, 4145-4153. (21) Weeks, K. M., and Crothers, D. M. (1991) RNA Recognition by Tat-Derived Peptides: Interaction in the Major Groove? Cell 66, 577-588. (22) Calnan, B. J., Tidor, B., Biancalana, S., Hudson, D., and Frankel, A. D. (1991) Arginine-Mediated RNA Recognition: The Arginine Fork. Science 252, 1167-1171. (23) Calnan, B. J., Biancalana, S., Hudson, D., and Frankel, A. D. (1991) Analysis of Arginine-Rich Peptides from the HIV Tat Protein Reveals Unusual Features of RNA Protein Recognition. Genes Dev. 5, 201-210. (24) Churcher, M. J., Lamont, C., Hamy, F., Dingwall, C., Green, S. M., Lowe, A. D., Butler, P. J. C., Gait, M. J., and Karn, J. (1993) High Affinity Binding of TAR RNA by the Human Immunodeficiency Virus Type-1 tat Protein Requires Base-pairs in the RNA Stem and Amino Acid Residues Flanking the Base Region. J. Mol. Biol. 230, 90-110. (25) Long, K. S., and Crothers, D. M. (1995) Interaction of human immunodeficiency virus type 1 Tat-derived peptides with TAR RNA. Biochemistry 34, 8885-8895. (26) Wang, Z., and Rana, T. M. (1996) RNA conformation in the Tat-TAR complex determined by site-specific photocrosslinking. Biochemistry 35, 6491-6499. (27) Huq, I., and Rana, T. M. (1997) Probing the proximity of the core domain of an HIV-1 Tat fragment in a Tat-TAR complex by affinity cleaving. Biochemistry 36, 12592-12599. (28) Wang, Z., Wang, X., and Rana, T. M. (1996) Protein orientation in the Tat-TAR complex determined by psoralen photocross-linking. J. Biol. Chem. 271, 16995-16998. (29) Tinoco, I., Jr., Puglisi, J. D., and Wyatt, J. R. (1990) RNA folding. Nucleic Acids Mol. Biol. 4, 205-226.

Wang et al. (30) Wyatt, J. R., Puglisi, J. D., and Tinoco, I., Jr. (1989) RNA folding: Pseudoknots, loops and bulges. Bioassays 11, 100106. (31) Shah, K., Wu, H., and Rana, T. M. (1994) Synthesis of uridine phosphoramidite analogues: Reagents for site-specific incorporation of photoreactive sites into RNA sequences. Bioconjugate Chem. 5, 508-512. (32) Wang, Z., and Rana, T. M. (1995) Chemical Conversion of a TAR RNA-Binding Fragment of HIV-1 Tat Protein into a Site-Specific Cross-linking Agent. J. Am. Chem. Soc. 117, 5438-5444. (33) Shah, K., Neenhold, H., Wang, Z., and Rana, T. M. (1996) Incorporation of an artificial protease and nuclease at the HIV-1 tat binding site of trans-activation response RNA. Bioconjugate Chem. 7, 283-289. (34) Ping, Y.-H., Liu, Y., Wang, X., Neenhold, H. R., and Rana, T. M. (1997) Dynamics of RNA-protein interactions in the HIV-1 Rev-RRE complex visualized by 6-thioguanosinemediated photo-cross-linking. RNA 3, 850-860. (35) Sumner-Smith, M., Roy, S., Barnett, R., Reid, L. S., Kuperman, R., Delling, U., and Sonenberg, N. (1991) Critical chemical features in trans-acting-responsive RNA are required for interaction with human immunodeficiency virus tye 1 tat protein. J. Virol. 65, 5196-5202. (36) Hamy, F., Asseline, U., Grasby, J., Iwai, S., Pritchard, C., Slim, G., Butler, P. J. G., Karn, J., and Gait, M. J. (1993) Hydrogen-bonding Contacts in the Major Groove are Required for Human Immunodeficiency Virus Type-1 tat Protein Recognition of TAR RNA. J. Mol. Biol. 230, 111-123. (37) Pritchard, C. E., Grasby, J. A., Hamy, F., Zacharek, A. M., Singh, M., Karn, J., and Gait, M. J. (1994) Methylphosphonate mapping of phosphate contacts critical for RNA recognition by the human immunodeficiency virus tat and rev proteins. Nucleic Acids Res. 22, 2592-2600. (38) Naryshkin, N. A., Farrow, M. A., Ivanovskaya, M. G., Oretskaya, T. S., Shabarova, Z. A., and Gait, M. J. (1997) Chemical cross-linking of the human immunodeficiency virus type 1 Tat protein to synthetic models of the RNA recognition sequence TAR containing site-specific trisubstituted pyrophosphate analogues. Biochemistry 36, 3496-3505. (39) Favre, A., and Fourrey, J.-L. (1995) Structural probing of small endonucleolytic ribozymes in solution using thiosubstituted nucleobases as intrinsic photolabels. Acc. Chem. Res. 28, 375-382. (40) Milligan, J. F., Groebe, D. R., Witherell, G. W., and Uhlenbeck, O. C. (1987) Oligoribinucleotide synthesis using T7 RNA polymerase and synthetic DNA templates. Nucleic Acids Res. 15, 8783-8798. (41) Neenhold, H. R., and Rana, T. M. (1995) Major Groove Opening at the HIV-1 Tat Binding Site of TAR RNA Evidenced by a Rhodium Probe. Biochemistry 34, 6303-6309. (42) King, D. S., Fields, C. G., and Fields, G. B. (1990) A cleavage method which minimizes side reactions following Fmoc solid-phase peptide synthesis. Int. J. Pept. Protein Res. 36, 255-266. (43) Cordingley, M. G., La Femina, R. L., Callahan, P. L., Condra, J. H., Sardana, V. V., Graham, D. J., Nguyen, T. M., Le Grow, K., Gotlib, L., Schlabach, A. J., and Colonno, R. J. (1990) Sequence-specific Interaction of Tat protein and Tat peptides with the Transactivation-responsive Sequence Element of Human Immunodeficiency Virus Type 1 in vitro. Proc. Natl. Acad. Sci. U.S.A. 87, 8985-8989. (44) Subramanian, T., Govindarajan, R., and Chinnadurai, G. (1991) Heterologous basic domain substitutions in the HIV-1 Tat protein reveal an arginine-rich motif required for transactivation. EMBO J. 10, 2311-2318. (45) Weeks, K. M., Ampe, C., Schultz, S. C., Steitz, T. A., and Crothers, D. M. (1990) Fragments of the HIV-1 Tat Protein Specifically Bind TAR RNA. Science 249, 1281-1285. (46) Harper, J. W., and Logsdon, N. J. (1991) Refolded HIV-1 Tat Protein Protects Both Bulge and Loop Nucleotides in TAR RNA from Ribonucleolytic Cleavage. Biochemistry 30, 80608066. (47) Delling, U., Reid, L. S., Barnett, R. W., Ma, M. Y.-X., Climie, S., Summer-Smith, M., and Sonenberg, N. (1992) Conserved Nucleotides in the TAR RNA stem of Human

Proximity of a Tat Peptide to TAR RNA Loop Region Immunodeficiency Virus Type 1 are Critical for Tat Binding and Transactivation: Model for TAR RNA Tertiary Structure. J. Virology 66, 3018-3025. (48) Tao, J., and Frankel, A. D. (1992) Specific binding of arginine to TAR RNA. Proc. Natl. Acad. Sci. U.S.A. 89, 27232726. (49) Wang, Z., and Rana, T. M. (1998) RNA-protein interactions in the Tat-trans-activation response element complex determined by site-specific photo-cross-linking. Biochemistry 37, 4235-4243. (50) Musier-Forsyth, K., and Schimmel, P. (1994) Acceptor helix interactions in a class II tRNA Synthetase: Photoaffinity cross-linking of an RNA minidulex substrate. Biochemistry 33, 773-779. (51) Farrow, M. A., Aboul-ela, F., Owen, D., Karpeisky, A., Beigelman, L., and Gait, M. J. (1998) Site-specific crosslinking of amino acids in the basic region of human immunodeficiency virus type 1 Tat peptide to chemically modified TAR RNA duplexes. Biochemistry 37, 3096-3108. (52) Aboul-ela, F., Karn, J., and Varani, G. (1995) The structure of the human immunodeficiency virus type-1 TAR RNA reveals principles of RNA recognition by Tat protein. J. Mol. Biol. 253, 313-332.

Bioconjugate Chem., Vol. 10, No. 3, 1999 519 (53) Bayer, P., Kraft, M., Ejchart, A., Westendorp, M., Frank, R., and Ro¨sch, P. (1995) Structural studies of HIV-1 Tat protein. J. Mol. Biol. 247, 529-535. (54) Feng, S., and Holland, E. C. (1988) HIV-1 tat transactivation requires the loop sequence within TAR. Nature 334, 165-167. (55) Berkhout, B., and Jeang, K.-T. (1989) trans activation of human immunodeficiency virus type 1 is sequence specific for both the single-stranded bulge and loop of the transacting-responsive hairpin: a quantitative analysis. J. Virol. 63, 5501-5504. (56) Wang, X., Huq, I., and Rana, T. M. (1997) HIV-1 TAR RNA recognition by an unnatural biopolymer. J. Am. Chem. Soc. 119, 6444-6445. (57) Wei, P., Garber, M. E., Fang, S.-M., Fischer, W. H., and Jones, K. A. (1998) A novel CDK9-associated C-type cyclin interacts directly with HIV-1 Tat and mediates its highaffinity, loop specific binding to TAR RNA. Cell 92, 451-462.

BC980145J