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Nucleic Acids Research Pages 1084-1093  

Visualizing tertiary folding of RNA and RNA-protein interactions by a tethered iron chelate: analysis of HIV-1 Tat-TAR complex
Introduction
Materials And Methods
   Buffers
   RNA synthesis
   Conjugation of an EDTA analog to TAR RNA
   Gel retardation assays
   RNA self-cleavage reactions
   Fe(II)-EDTA footprinting
   RNA sequencing
Results
   Site-specific labeling of RNA with EDTA
   Determination of dissociation constants
   Site-specific cleavage of TAR RNA
   Tat-TAR interactions
Discussion
Acknowledgements
References

Visualizing tertiary folding of RNA and RNA-protein interactions by a tethered iron chelate: analysis of HIV-1 Tat-TAR complex

Visualizing tertiary folding of RNA and RNA-protein interactions by a tethered iron chelate: analysis of HIV-1 Tat-TAR complex

Ikramul Huq, Natarajan Tamilarasu and Tariq M. Rana*

Department of Pharmacology, Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854, USA

Received October 1, 1998; Revised November 30, 1998; Accepted December 21, 1998

ABSTRACT

Replication of human immunodeficiency virus type 1 (HIV-1) requires specific interactions of Tat protein with the trans-activation responsive region (TAR) RNA, a 59 base stem-loop structure located at the 5[prime]-end of all HIV transcripts. We have used an intramolecular RNA self-cleaving strategy to determine the folding of TAR RNA and its interactions with a Tat peptide. We incor-porated an EDTA analog at position 24 in the HIV-1 Tat binding site of the TAR RNA. After isolation and purification of the EDTA-TAR conjugate, RNA self-cleavage was initiated by the addition of an iron salt, ascorbate and hydrogen peroxide. Hydroxyl radicals generated from the tethered Fe(II) cleaved TAR RNA backbone in two localized regions. Sites of RNA cleavage were mapped by sequencing reactions. A Tat fragment, Tat(38-72), specifically inhibited RNA self-cleavage. To determine the structural changes caused by the Tat peptide, we performed Fe(II)-EDTA footprinting experiments on Tat-TAR complex. Our high-resolution footprinting results suggest that the inhibition of self-cleavage of EDTA-TAR is due to two effects of Tat binding: (i) Tat binds in the bulge and protects residues in the vicinity of the bulge from self-cleavage and (ii) RNA goes through a structural change where EDTA-U24 is rigidly positioned out of the helix and cannot get access to other nucleotides in the loop of TAR RNA, which are not protected by the Tat peptide. Our results demonstrate that Fe(II)-EDTA-mediated RNA self-cleavage can be applied to study RNA tertiary structures and RNA-protein interactions.

INTRODUCTION

RNA molecules fold into well-defined three-dimensional structures to perform biological and chemical functions (1,2). Folded structures of RNA contain regions of double-stranded duplex, hairpins, internal loops, bulged bases and pseudoknotted structures (3). Knowledge of the three-dimensional structure and general rules for RNA folding will be valuable to infer a more detailed mechanism of RNA function. The RNA structure field has developed rapidly and a number of RNA structures have been determined by X-ray crystallographic and NMR analysis (4). In the absence of high-resolution data, new biochemical methods are needed to analyze the folded structure of RNA and ribonucleoprotein complexes.

An iron-EDTA complex cleaves DNA non-specifically and can be applied to determine the helical periodicity of DNA and structural details of bent DNA (5,6). In the presence of ascorbate and H2O2, Fe(II)-EDTA complex generates hydroxyl radicals which are responsible for DNA scission (7). Attachment of an Fe(II)-EDTA complex to small molecules, proteins and oligonucleotides has proven to be a useful tool for analyzing the structures of such molecules in complex with DNA or RNA (8-17). Covalent attachment of Fe(II)-EDTA at a specific site in a nucleic acid structure localizes the generation of hydroxyl radical species which cleaves at several nucleotides positioned in close proximity to the metal chelate. Cleavage fragments can be separated by high-resolution polyacrylamide gel electrophoresis. The size and amount of cleaved fragments relate to the distance in the three-dimensional structure. Therefore, structural information regarding the nucleotides located in the nearest neighborhood of uniquely placed Fe(II)-EDTA can be obtained.

The promoter of the human immunodeficiency virus type 1 (HIV-1), located in the U3 region of the viral long terminal repeat (LTR), is an inducible promoter which can be stimulated by the trans-activator protein, Tat (18). As in other lentiviruses, Tat protein is essential for trans-activation of viral gene expression (19-23). HIV-1 Tat protein acts by binding to the trans-activation responsive (TAR) RNA element, a 59 base stem-loop structure located at the 5[prime] end of all nascent HIV-1 transcripts (24). Upon binding to the TAR RNA sequence, Tat causes a substantial increase in transcript levels (25-29). The increased efficiency in transcription may result from preventing premature termination of the transcriptional elongation complex (30-34). TAR RNA was originally localized to nucleotides +1 to +80 within the viral LTR (35). 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 (36-38). TAR RNA contains a 6 nt loop and a 3 nt pyrimidine bulge which separates two helical stem regions (24,27,35,36). The trinucleotide bulge is essential for high affinity and specific binding of the Tat protein (39,40). The loop region is required for in vivo trans-activation but is not involved in Tat binding (40-44).

Here we report the application of Fe(II)-EDTA-mediated RNA self-cleavage to probe folding of HIV-1 TAR RNA and its interactions with Tat protein. We incorporated an EDTA analog at the C24 position in the trinucleotide bulge of TAR RNA. Measurements of the binding constant (Kd) for wild-type TAR-Tat and EDTA modified TAR-Tat indicated that the folded structure of TAR RNA was functional and not altered significantly by the presence of EDTA analog at the C24 position. EDTA modified TAR RNA was self-cleaved at specific positions upon addition of Fe(II) salt, ascorbate and H2O2. RNA self-cleavage was monitored in the presence and absence of a Tat fragment. Sites of RNA cleavage were mapped by sequencing reactions, revealing the proximity of the Fe(II)-EDTA moiety in TAR RNA structure. Our results show that Fe(II)-EDTA-mediated RNA self-cleavage can be applied to study RNA tertiary structures.

MATERIALS AND METHODS

Buffers

All buffer pH values refer to measurements at room temperature. TK buffer: 50 mM Tris-HCl (pH 7.4), 20 mM KCI, 0.1% Triton X-100. Transcription buffer: 40 mM Tris-HCl (pH 8.1), 1 mM spermidine, 0.01% Triton X-100, 5 mM DTT. TBE buffer: 45 mM Tris-borate, pH 8.0, 1 mM EDTA. Sample loading buffer: 9 M urea, 1 mM EDTA and 0.1% bromophenol blue in1× TBE buffer. Hydrolysis buffer: 50 mM Na2CO3/NaHCO3, pH 9.2. Elution buffer: 1× TBE and 10% sodium acetate (3 M), pH 5.5. Digestion buffer: 100 mM Tris-HCl (pH 7.8), 10 mM CaCl2.

RNA synthesis

RNAs were synthesized by chemical and enzymatic methods. Modified TAR RNA was synthesized on an Applied Biosystems Model 392 DNA/RNA synthesizer using 2-cyanoethyl phosphor-amidite chemistry. All the monomers of (2-cyanoethyl)phosphor-amidites were obtained from Glen Research (Sterling, Virginia). Phosphoramidite 1 (0.15 M solution in CH3CN) was used to incorporate a modified uridine at position 24 in the TAR RNA sequence. Synthesis of the phosphoramidite 1 was accomplished according to published methods (45,46). RNA (1 µmol) containing nucleoside 1 was deprotected by treatment with NH3 saturated methanol (2 ml) at 25°C for 17 h. Product was filtered and dried in Speedvac. The pellet was dissolved in 0.5 ml of 50% TEA:3HF [in dimethyl sulfoxide (DMSO)] solution and left at room temperature for 16 h. Deprotected RNA was precipitated by the addition of 2 ml of isopropyl alcohol. After deprotection, RNA was purified and characterized as described earlier by Shah et al. (46).

Wild-type and mutant TAR RNAs were prepared by in vitro transcription (47,48). All DNAs were synthesized on an Applied Biosystems ABI 392 DNA/RNA synthesizer. The template strands encode the sequences for wild-type and mutant TAR RNAs. The top strand is a short piece of DNA complementary to the 3[prime] end of all template DNAs having the sequence 5[prime]-TAATACGACTCACTATAG-3[prime]. 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 (20 µl) containing 8.0 pmol template DNA, 40-60 U 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 and stored in DEPC water at -20°C.

Enzymatically transcribed RNAs were 5[prime] dephosphorylated by incubation with calf intestinal alkaline phophatase (Promega) for 1 h at 37°C in 50 mM Tris-HCl (pH 9.0), 1 mM MgCl2, 0.1 mM ZnCl2, 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. Chemically synthesized RNA contains free 5[prime]-OH groups and does not require dephosphorylation procedures. The RNAs were 5[prime]-end-labeled with 0.5 µM [[gamma]-32P]ATP (6000 Ci/mmol) (ICN) per 100 pmol RNA by incubating with 16 U T4 polynucleotide kinase (New England Biolabs) in 70 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 5 mM DTT (48,49). The RNAs were labeled at the 3[prime]-end by ligation to cytidine 3[prime],5[prime]-[5[prime]-32P]bisphosphate ([32P]pCp) using T4 RNA ligase. Reaction mixtures (50 µl) contained 250 pmol RNA, 65 µCi [32P]pCp (3000 Ci/mmol; NEN[trade], Boston, MA) and 40 U T4 RNA ligase (New England Biolabs) in a buffer containing 50 mM Tris-HCl (pH 8.0), 3 mM DTT, 10 mM MgCl2, 25 mM NaCl, 50 mM ATP, 25 µg/ml bovine serum albumin (BSA) and 10% DMSO (v/v). After incubation at 4°C overnight, the labeled RNAs were purified by phenol-chloroform extraction and ethanol precipitation. 3[prime]- and 5[prime]-end-labeled RNAs were gel purified on a denaturing gel, visualized by autoradiography, eluted out of the gels, and desalted on a reverser-phase cartridge. The sequence of RNAs was determined by base hydrolysis and nuclease digestion.


Scheme 1. Site-specific EDTA labeling of TAR RNA.

Conjugation of an EDTA analog to TAR RNA

We used a bifunctional chelating agent (CITC) to label primary amino groups at position 24 in TAR RNA. Experimental outline and the structure of CITC are shown in Scheme 1. Synthesis of CITC was accomplished as described by Meares et al. (50). For conjugation reaction, ~250 pmol 3[prime]- or 5[prime]-32P-end-labeled NH2-TAR was mixed with 500 pmol of CITC in 0.1 M phosphate buffer (pH 9.0) and incubated at 37°C for 2-12 h. Conjugation reaction was stopped by adding an equal volume of 2× sample loading buffer. CITC-labeled TAR RNA was separated from unconjugated RNA on 20% acrylamide-8 M urea denaturing gels. Efficiencies of the conjugation reaction were determined by a PhosphorImager analysis (Molecular Dynamics). RNA bands corresponding to CITC-TAR conjugate were visualized by autoradiography, excised from the gel and eluted in 50 mM Tris, 16 mM boric acid, 1 mM EDTA and 0.5 M sodium acetate. CITC-TAR RNA was ethanol precipitated and resuspended in DEPC treated water for further studies.


Figure 1. (A) Secondary structure of TAR RNA used in this study. TAR RNA spans the minimal sequences that are required for Tat responsiveness in vivo (36) 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. Numbering of nucleotides in the RNA corresponds to their positions in wild-type TAR RNA. C24, site for incorporation of EDTA, is highlighted. (B) The structure of a modified uridine phosphoramidite 1.

Gel retardation assays

RNA-protein binding reactions (20 µl) contained 0.05 µM 5[prime]-32P-end-labeled RNA and increasing amounts of Tat(38-72) peptide from 0.1 to 1.0 µM. 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 non-denaturing 8% polyacrylamide gels containing 0.1% Triton X-100. Gels were run in a cold room at 300 V for 2 h. The relative amounts of free and bound RNA were determined by phosphorimaging.

RNA self-cleavage reactions

A typical cleavage reaction mixture (10 µl) contained 3[prime]- or 5[prime]-end-labeled EDTA-TAR RNA (50 nM) in a buffer containing 50 mM Tris-HCl (pH 7.4) and 20 mM KCl. The cleavage reaction was initiated by the addition of Fe(NH4)2(SO4)2·6H2O (40 nM) followed by sodium ascorbate (4 mM) and H2O2 (0.12%). Cleavage reaction in the presence of Tat and BSA contained 0.2 µM Tat(38-72) peptide and 5 µM BSA, respectively. The cleavage reactions were allowed to proceed for 5 min at room temperature, quenched by the addition of 10 µl of 2× sample loading buffer, and samples were loaded onto a 20% denaturing polyacrylamide gel. Cleavage products were visualized by a PhosphorImage analysis (Molecular Dynamics).

Fe(II)-EDTA footprinting

All reactions were carried out at room temperature and in a volume of 10 µl. Tat(38-72) peptide or water (for reference lanes) was added to an assay buffer. The final concentrations in an assay were 5 nM 3[prime]- or 5[prime]-end-labeled TAR RNA, 20 nM Tat peptide, 50 mM Tris-HCl (pH 7.4), 20 mM KCl and 0.1% Triton X-100. The solutions were allowed to equilibrate for 1 h. EDTA-Fe(II) solution was prepared by adding 2 mM freshly prepared ferrous ammonium sulfate [Fe(NH4)2(SO4)2·6H2O] to a solution of 4 mM EDTA. The freshly prepared EDTA-Fe(II) solution (2 mM) was added to the equilibrated RNA, and the reactions were allowed to equilibrate for 5 min. Cleavage reaction was initiated by the addition of 10 mM sodium ascorbate and 1% H2O2 and allowed to proceed for 1-10 min. The cleavage reaction was quenched by ethanol precipitation. Samples were dissolved in 10 µl of sample loading buffer, heated at 85°C for 2 min and immediately loaded onto a 20% denaturing polyacrylamide gel. Cleavage products were visualized by autoradiography and phosphorimage analysis.

RNA sequencing

Alkaline hydrolysis of RNAs was carried out in hydrolysis buffer for 8-12 min at 85°C. All ribonucleases were purchased from Pharmacia. RNAs were incubated with 0.1 U RNAse from Bacillus cereus per pmol 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. For RNase T1 digestion, RNAs were incubated with 0.05 U RNase per pmol RNA for 10 min at 37°C in 40 mM Tris-HCl (pH 8.0), 1 mM EDTA, 40 µg yeast tRNA. T1 cuts RNA at G nucleotides. Sequencing products were resolved on 20% denaturing gels and visualized by phosphorimage analysis.

RESULTS

Site-specific labeling of RNA with EDTA

The experimental strategy for site-specific chelate conjugation of TAR RNA is outlined in Scheme 1. A modified uridine phosphoramidite, compound 1 (Fig. 1), was synthesized according to previously published methods (45,46) and used to incorporate a reactive primary amino group at position 24 in TAR RNA sequence during chemical synthesis. Modified phosphoramidite was incorporated into RNA oligomers with >97% coupling efficiencies. RNA containing modified nucleoside was cleaved from the support and deprotected in NH3 saturated methanol at 25°C for 17 h. The primary amino group linked to position five of uridine-alkylamine (compound 1, Fig. 1) is revealed for modification by treatment with ammonia/methanol (45). After deprotection, RNA was purified and characterized as described earlier by Shah et al. (46).


Figure 2. (A) Conjugation reaction of TAR RNA with isothiocyanato chelate, CITC. Lanes 1-7, the conjugation reactions were carried out at 37°C for 0, 0.5, 1, 2, 4, 7 and 22 h, respectively. The reaction products are labeled as EDTA-TAR. (B) Quantitative analysis of conjugation reactions. The fraction of RNA in the EDTA-TAR conjugate was determined by phosphorimage analysis as described in Materials and Methods.

A bifunctional chelating agent (CITC) was used for site-specific EDTA labeling of the amino-modified TAR RNA duplex. Experimental outline and the structure of CITC are shown in Scheme 11. Bifunctional chelating agents are compounds that comprise both a strong metal-chelating group and a chemically reactive functional group. Synthesis of CITC was accomplished as described by Meares et al. (50). Conjugation reaction of CITC and amino-modified RNA was carried out at pH 9.0 at 37°C for different periods of time. Products of the conjugation reactions were analyzed on 8 M urea-20% polyacrylamide gels. As shown in Figure 2A, CITC-TAR conjugate migrates with lower electrophoretic mobility on a denaturing polyacrylamide gel. A control reaction between wild-type TAR and CITC gave no slow migrating products (data not shown). Quantification of the conjugation reaction and a kinetic analysis revealed that the reaction was completed in 4 h giving ~60% yields of the EDTA-RNA conjugate (Fig. 2B). Further incubation of the reaction mixtures did not significantly increase the formation of EDTA-TAR conjugate. However, longer time incubation at pH 9.0 and 37°C resulted in hydrolysis of RNA (Fig. 2A, lane 7). Incorporation of CITC in TAR RNA sequence was confirmed by enzymatic digestion and RNA sequencing. The hydrolysis ladder of the CITC-TAR conjugate is identical to that of unmodified control RNA up to the point of modification, C24. The hydrolysis fragments from the modified RNA are non-linear and migrate anomalously slowly, introducing a gap in the ladder relative to the unmodified control RNA. There is a clear gel mobility shift in the hydrolysis ladder of RNA after U23, indicating that U24 is covalently modified with EDTA analog (Fig. 4, lane 2). Base hydrolysis of unmodified TAR RNA showed no gaps in these positions including U24 in the sequence (data not shown). Since there were no detectable amounts of unmodified U24 present in Figure 4 (lane 2), we conclude that the EDTA-TAR conjugate was stable to alkaline hydrolysis conditions.


Figure 3. Binding of Tat(38-72) peptide to TAR RNA (A) or Fe(II)-EDTA-TAR conjugate (B). Binding reactions contained 50 nM of 5[prime]-32P-end-labeled RNA and increasing concentrations of Tat peptide as described in Materials and Methods. Lane 1 was a control lane without the peptide. RNA-peptide complexes are shown as R-P.

Determination of dissociation constants

To further characterize and evaluate the Tat-binding capabilities of EDTA-TAR conjugate, we determined the dissociation constants for Tat(38-72) peptide and compared with the wild-type TAR RNA. Equilibrium dissociation constants of the Tat(38-72)-TAR complexes were measured using direct electrophoretic mobility assays (17,46,51-53). For direct mobility shift assays, the fractional saturation of 50 nM 5[prime]-32P-end-labeled TAR RNA or EDTA-TAR RNA was measured as a function of Tat(38-72) peptide. A typical gel of these experiments is shown in Figure 3. Dissociation constants were calculated from multiple sets of experiments which showed that the wild-type TAR RNA and EDTA-TAR RNA bind Tat(38-72) with a Kd of 0.15 ± 0.05 and 0.19 ± 0.07 µM, respectively. A relative dissociation constant (Krel) can be determined by measuring the ratios of wild-type TAR RNA to the EDTA-TAR RNA Kd for Tat. Our results demonstrate that the calculated value for Krel was 0.79, indicating that the metal chelate attachment did not significantly alter the Tat binding affinities of TAR RNA.


Figure 4. Cleavage of 5[prime]-32P-labeled EDTA-TAR conjugate. Lane 1, B.cereus ladder of RNA; lane 2, alkaline hydrolysis of RNA; lane 3, RNA without the addition of iron salt, ascorbate and H2O2; lane 4, RNA subjected to self-cleavage reaction by the addition of iron salt, ascorbate and H2O2; lane 5, RNA self-cleavage reaction in the presence of BSA; lane 6, RNA self-cleavage reaction in the presence of Tat(38-72) peptide; lane 7, RNA self-cleavage reaction in the absence of ferrous ammonium sulfate [Fe(NH4)2(SO4)2·6H2O]. Major sites of cleavage are marked.

   A
   B

Figure 5. (A) Cleavage of 3[prime]-32P-labeled EDTA-TAR conjugate. Lane 1, B.cereus ladder of RNA; lane 2, hydrolysis ladder of RNA; lane 3, RNA without the addition of iron salt, ascorbate and H2O2; lane 4, RNA self-cleavage reaction in the absence of ferrous ammonium sulfate; lanes 5 and 6, RNA self-cleavage reactions by the addition of 20 and 40 µM iron salt, respectively; lane 7, RNA self-cleavage reactions in the presence of Tat(38-72) peptide. Major sites of cleavage are marked. (B) Three-dimensional representation of the RNA self-cleavage method using a tethered iron chelate to probe tertiary folding of TAR RNA. Iron(II) is covalently attached at a specific site in the RNA sequence and arrows show the regions of RNA cleaved by the production of localized hydroxyl radicals.

Site-specific cleavage of TAR RNA

EDTA-modified TAR RNA was 5[prime]-end-labeled with 32P, and metal-catalyzed cleavage reactions were performed as described in Materials and Methods. Cleavage products were separated by denaturing polyacrylamide gel electrophoresis. Results of these experiments are shown in Figure 4. The cleavage sites in TAR RNA were assigned by comparison with 5[prime]-end-labeled products of alkaline hydrolysis, random Fe-EDTA cleavage ladder of the RNA, and ribonuclease B.cereus and T1 reactions. As shown in Figure 4, cleavage by the Fe-EDTA chelate specifically attached at position 24 in TAR RNA was not random and a few specific sites were cleaved. Specific cleavage of TAR RNA was observed at the bulge residues (C24, U25 and G26), in the loop region (G33, G34 and A35), and at the strand opposite to the bulge (U38 and C39). Intensities of the cleavage product bands on the gel showed that C24 was cleaved most efficiently, as expected, while other nucleotides were cleaved with lower yields. Control experiments showed that the EDTA-TAR RNA cleavage was intramolecular. The RNA cleavage reaction was independent of the EDTA-TAR concentration, indicating an intramolecular cleavage reaction (data not shown).

Figure 4 shows that no detectable cleavage of TAR RNA was observed when the RNA was subjected to cleavage reaction conditions in the absence of iron salt (lane 7). Lane 3 shows that TAR RNA was stable during experimental conditions and no background RNA degradation was detected. Further control experiments established that free Fe(II)-EDTA (40 µM) did not cleave TAR RNA under standard cleavage conditions (data not shown). These results indicate that Fe-EDTA chelate attached to the uridine 24 of TAR RNA lies in close proximity to C25, G26, U38 and C39 in the bulge region and three nucleotides (G33, G34 and A35) from the loop sequence also come in contact with the iron chelate.

Although Figure 4 shows cleavage at C24 position clearly, RNA fragments containing Fe-EDTA groups migrate anomalously making characterization of other cleavage sites difficult. To clearly identify the cleavage sites in the loop and the strand opposite to the bulge, we labeled EDTA-TAR RNA at its 3[prime]-end and subjected to self-cleavage reactions. As shown in Figure 5, cleavage fragments up to the bulge region were clearly resolved and the cleavage sites can be identified unambiguously. Figure 5 (lanes 5 and 6) shows that cleavage occurred at three nucleotides in the loop region (G33, G34 and A35) and at two nucleotides in the strand opposite to the bulge sequence (U38 and C39). Control experiments showed that the 3[prime]-end-labeled EDTA-TAR was stable (lane 3) and no detectable cleavage occurred in the absence of iron salt (lane 4). Therefore, results of Figures 4 and 5 demonstrate that tethered Fe-EDTA chelate cleaved TAR RNA at the trinucleotide bulge and loop regions.


Figure 6. Fe(II)-EDTA footprinting experiment on the 5[prime]-32P-labeled wild-type TAR RNA. Lane 1, Fe(II)-EDTA cleavage ladder of RNA; lanes 2-5, Fe(II)-EDTA cleavage of RNA-Tat(38-72) complex for 1, 2, 5 and 10 min, respectively; lanes 6 and 7, RNA and RNA-Tat(38-72) complex without the cleavage reactions, respectively. Nucleotides protected from cleavage in the presence of Tat(38-72) are marked.

Tat-TAR interactions

To determine the effect of Tat on RNA self-cleavage, we performed cleavage reactions in the presence of Tat(38-72), which binds TAR RNA with high specificity and includes the basic and part of the core regions of Tat protein (53). As shown in Figures 4 (lane 6) and 5 (lane 7), only C24 was cleaved in the presence of Tat(38-72) peptide and cleavage at other nucleotides was inhibited. Addition of BSA did not inhibit the RNA self-cleavage reactions (Fig. 4, lane 5) indicating that Tat specifically inhibits TAR RNA self-cleavage. HIV-1 Rev protein that binds Rev response element RNA also had no inhibitory effect on the self-cleavage of TAR RNA (data not shown). There could be three possible reasons for the inhibition of EDTA-TAR self-cleavage in the Tat-TAR complex: (i) Tat binding causes a structural change in RNA and moves the tethered Fe-EDTA away from the TAR structure; (ii) Tat binds to the RNA and protects specific residues from cleavage; and (iii) Tat causes structural change in the RNA as well as protects specific residues from cleavage. To resolve these questions, we performed Fe(II)-EDTA footprinting experiments on the Tat-TAR complex (Figs 6 and 7). Fe(II)-EDTA footprinting experiments [50 mM Tris-HCl (pH 7.4), 20 mM KCl and 0.1% Triton X-100] on 5[prime]- and 3[prime]-end-labeled TAR RNA revealed that Tat(38-72) protects nucleotides in the bulge as well as below and above the bulge. Nucleotides G21, A22, U23, U25 and G26 were protected from cleavage when 5[prime]-end-labeled TAR RNA was used in the footprinting reaction (Fig. 6). Cleavage protection was observed at C37, U38 and C39 residues when 3[prime]-end-labeled TAR RNA was subjected to footprinting reactions (Fig. 7). Addition of BSA did not protect any regions of TAR RNA indicating that Tat protection was a specific effect (Fig. 7, lane 5). C24 in the bulge and nucleotides in the loop sequence were not protected by Tat(38-72). Inhibition of RNA self-cleavage by Tat in the bulge region can be explained by a protection effect. Since the loop sequence was not protected by Tat, it cannot be ruled out that RNA structure is changed to position U24-EDTA away from the loop residues. These results suggest that the inhibition of EDTA-TAR RNA self-cleavage in the presence of Tat(38-72) is due to cleavage protection and a structural change in RNA.


Figure 7. Fe(II)-EDTA footprinting experiment on the 3[prime]-32P-labeled wild-type TAR RNA. Lane 1, RNA in the absence of cleavage reagents; lane 2, Fe(II)-EDTA cleavage ladder of RNA; lanes 3 and 4, Fe(II)-EDTA cleavage of RNA-Tat(38-72) complex in the presence of 20 and 40 nM Tat(38-72), respectively; lane 5, Fe(II)-EDTA cleavage of RNA in the presence of BSA. Nucleotides protected from cleavage in the presence of Tat(38-72) are marked.

   A
   B

Figure 8. (A) (left) [U24-EDTA]TAR RNA self-cleavage sites shown in a tertiary structure of HIV-1 TAR RNA (57). Nucleotide C24 that is replaced with U-EDTA is shown in green. Nucleotides cleaved by the tethered Fe(II)-EDTA are shown: U25 and G26 (magenta); G33, G34 and A35 (white); U38 and C39 (cyan). Ribbon structure of TAR RNA is shown as yellow lines and nucleotides in red. (Right) Proposed folding of TAR RNA loop sequence showing that loop residues are located in the vicinity of the bulge region in RNA tertiary structure. The RNA structure is derived from F.Aboul-ela et al. (57) and RNA is folded to bring all the cleaved residues in the proximity of the iron chelate. For clarity, only C24 and cleaved nucleotides are shown. (B) Summary of Fe(II)-EDTA footprinting experiments on TAR RNA. A view of TAR RNA structure in the presence of l-argininamide (60,62) displaying nucleotides protected from Fe(II)-EDTA cleavage in the presence of Tat(38-72) and two nucleotides, C24 and U40 (red), which are not protected. G21, A22, U23, U25 and G26 are shown in green and C37, U38 and C39 are shown in cyan. Ribbon structure of TAR RNA is shown as yellow lines. Structures of RNA were visualized using Insight II software on an IRIS work station.

DISCUSSION

We have used a metal-catalyzed RNA self-cleavage strategy to determine the tertiary folding of TAR RNA and its interactions with a Tat fragment. Our results show that, in solution, an Fe-EDTA attached at the U24 position of TAR RNA cleaves the RNA in the bulge region and at three nucleotides from the loop sequence. Self-cleavage of TAR RNA was inhibited in the presence of Tat(38-72) peptide.

To introduce a site-specific metal-binding site in TAR RNA sequence, we synthesized a modified uridine phosphoramidite (compound 1, Fig. 1) and used it to substitute for C24 of TAR RNA by chemical synthesis. We chose the C24 position because this residue lies in the trinucleotide bulge region and is in close proximity to U23, an essential residue. Furthermore, C24 is not essential for TAR function and can be changed without affecting Tat-binding (54). During cleavage and deprotection of chemically synthesized RNA, the primary amino group linked to the five position of uridine-alkylamine was exposed and was chemically modified with an EDTA analog (CITC). Kinetic analysis of the chemical modification reaction showed that the reaction was completed in 4 h giving ~60% yields of the EDTA-RNA conjugate (Fig. 2B). In a recent report (46), we synthesized TAR RNA containing a primary amine linked to the five position of uridine by an ethyl propionamide linker and labeled it with CITC. The yields of conjugation reactions between uridine-5-aminoethyl propionamide and CITC were ~20% (46). It is interesting to note that deletion of an amide bond from the linker between the amino group and uridine significantly increases the efficiency of chemical modification of the amino group. This can be explained by the rigidity of the amide bond which is not present in the alkylamine linker. Our results therefore suggest that flexible linkers should be employed to introduce reactive groups in nucleic acid structures for further chemical modifications with high efficiencies.

Attachment of an Fe(II)-EDTA in the bulge region of TAR RNA localizes the production of hydroxyl radicals generated by the addition of ascorbate and hydrogen peroxide. Hydroxyl radical species are short lived and the cleavage of nucleic acids is observed within a 10 Å radius from the site of production of these radicals (5,55). Recently, Tullius and co-workers demonstrated that the hydroxyl radical reacts with the various hydrogen atoms of the deoxyribose in the order 5[prime] H > 4[prime] H > 3[prime] H [ap] 2[prime] H [ap] 1[prime] H (56). We observed the cleavage of RNA backbone in the range of ~5-20 Å indicating that these nucleotides are within the reach of Fe(II)-EDTA tethered to the modified U24 in TAR RNA. The strongest cleavage was observed at U24 while cleavage reactions at other nucleotides were less efficient. Cleavage results in the context of three-dimensional TAR RNA structure are shown in Figure 8. We measured the distances from C5 (site of EDTA attachment in substituted U) of C24 to the preferred site of hydrogen abstraction, C5[prime] of ribose, of different nucleotides in a free TAR RNA structure determined by NMR (57). The distances between C5 of C24 and C5[prime] of U25, G26, G33, G34, A35, U38 and C39 were 8.6, 10.7, 20.6, 15.5, 16.5, 19.5 and 18.3 Å, respectively. Major cleavage sites are located in the vicinity of the trinucleotide bulge region. However, it is interesting to note that three nucleotides from the loop were also cleaved while other nucleotides in the same distance range were not significantly cleaved. For example, a very minor cleavage was detected at A22, G21 and A20. We propose two explanations for this result. (i) Dynamics of TAR RNA structure bring certain residues in the vicinity of the Fe(II)-EDTA complex. For example, the hairpin loop of TAR RNA has a flexible structure in solution, and there is no compelling evidence that the loop is closed by any non-Watson-Crick pairs, such as C.AH+ or G.U base pairs (58). This dynamic nature of the loop may form structured regions transiently, and loop residues may come in close proximity to the metal chelate. (ii) Every ribose at fixed distance does not have identical reactivity towards hydroxyl radicals generated by a tethered chelate. The RNA backbone in the loop region could be exposed to the solvent making it a preferred position for Fe(II)-EDTA interactions. We propose that TAR RNA loop sequence has a dynamic structure and loop residues are located in the vicinity of the bulge region in RNA tertiary structure for part of the time (Fig. 8).

In the presence of a Tat fragment, Tat(38-72), RNA self-cleavage occurs only at C24 residue (Fig. 4, lane 6). This effect of Tat on inhibition of TAR RNA self-cleavage was specific because the addition of BSA did not quench the RNA cleavage reaction. Previous studies show that Tat protein contacts TAR RNA in a widened major groove (48,49,59). One possibility is that when Tat occupies the major groove of RNA in the bulge region, it abolishes the access of Fe(II)-EDTA moiety to the other residues except U24. It is also quite possible that Tat initiates contacts with TAR RNA in the major groove causing RNA to undergo a structural change where EDTA-U24 is rigidly positioned and cannot get access to other loop or bulge residues. To address this question, we performed Fe(II)-EDTA footprinting experiments on the Tat-TAR complex (Figs 6 and 7). In the presence of Tat(38-72), cleavage protection was observed at G21, A22, U23, U25, G26, C37, U38 and C39 residues in TAR RNA. On the basis of these results, we propose that the inhibition of self-cleavage of EDTA-TAR is due to two effects of Tat binding: (i) Tat binds in the bulge and protects residues in the vicinity of the bulge from self-cleavage; and (ii) RNA goes through a structural change where EDTA-U24 is rigidly positioned out of the helix and cannot access other nucleotides in the loop of TAR RNA, which are not protected by the Tat peptide. A model summarizing footprinting results and proposed structural changes in TAR RNA is shown in Figure 8.

Footprinting pattern for Tat(38-72) peptide is mostly consistent with a binding site that includes bulge residues. These results also provide very interesting new information for Tat-TAR recognition. 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 (44,53). In addition to the trinucleotide bulge region, two base pairs above and below the bulge also contribute significantly to Tat binding (53,59). A base pair above the bulge, A27:U38, is involved in base-triple formation and is essential for Tat binding (60). Our high-resolution Fe(II)-EDTA footprinting results indicate that A27 is not protected and U25 is protected from cleavage in a Tat-TAR complex (Fig. 6). However, U38 is clearly protected from Fe-EDTA cleavage in the presence of Tat peptide (Fig. 7). Since hydroxyl radicals cleave RNA via hydrogen abstraction from the sugar moiety, our results indicate that the ribose of U25 is protected from cleavage while the ribose of A27 is exposed to the solvent in a Tat-TAR complex. Another interesting result from footprinting experiments is that C24 and U40 are not protected from cleavage in the presence of Tat peptide. This result is in complete agreement with our previous photocrosslinking studies showing that C24 and U40 are not essential for Tat recognition because Tat was able to bind a TAR RNA structure containing an interrupted bulge formed by a covalent link between U40 and C24 (48).

Our results demonstrate that RNA self-cleavage can be used to study RNA folding and RNA-protein interactions. We have used a small well-characterized RNA-protein complex in our experiments and shown the application of this approach. Our cleavage results correlate very well with mutagenesis and NMR studies and also provide additional information about dynamics of RNA folding and RNA-protein interactions. This technique can easily be extended to study larger RNA structures. Small RNAs containing primary amino groups can be chemically synthesized and labeled site-specifically with EDTA analogs. Site-specifically modified RNA molecules can be ligated into longer pieces of RNA with the use of bacteriophage T4 DNA ligase (61). This approach can provide a useful tool to study tertiary folding of larger RNAs and ribonucleoprotein complexes.

ACKNOWLEDGEMENTS

We thank Xilu Wang for RNA synthesis, Dr Fareed Aboul-ela for NMR coordinates for TAR RNA, and Dr Prem Yadav for his help in computer modeling. This work was supported in part by the National Institutes of Health Grants AI 41404, TW00702 and a Research Career Development Award to T.M.R.

REFERENCES

1. Draper,D.E. (1996) Trends Biochem. Sci., 21, 145-149. MEDLINE Abstract

2. Tanner,M.A. and Cech,T.R. (1997) Science, 275, 847-849. MEDLINE Abstract

3. Tinoco,I.,Jr, Puglisi,J.D. and Wyatt,J.R. (1990) Nucleic Acids Res. Mol. Biol., 4, 205-226.

4. Uhlenbeck,O.C., Pardi,A. and Feigon,J. (1997) Cell, 90, 833-840. MEDLINE Abstract

5. Tullius,T.D. and Dombroski,B.A. (1985) Science, 230, 679-681. MEDLINE Abstract

6. Price,M.A. and Tullius,T.D. (1992) Methods Enzymol., 212, 194-219. MEDLINE Abstract

7. Pogozelski,W.K., McNeese,T.J. and Tullius,T.D. (1995) J. Am. Chem. Soc., 117, 6428-6433.

8. Dervan,P.B. (1986) Science, 232, 464-471. MEDLINE Abstract

9. Moser,H.E. and Dervan,P.B. (1987) Science, 238, 645-650. MEDLINE Abstract

10. Sluka,J.P., Horvath,S.J., Bruist,M.F., Simon,M.I. and Dervan,P.B. (1987) Science, 238, 1129-1132. MEDLINE Abstract

11. Ebright,Y.W., Chen,Y., Pendergrast,P.S. and Ebright,R.H. (1992) Biochemistry, 31, 10664-10670. MEDLINE Abstract

12. Murakami,K., Kimura,M., Owens,J.T., Meares,C.F. and Ishihama,A. (1997) Proc. Natl Acad. Sci. USA, 94, 1709-1714. MEDLINE Abstract

13. Wang,J.-F. and Cech,T.R. (1992) Science, 256, 526-529. MEDLINE Abstract

14. Han,H. and Dervan,P.B. (1994) Proc. Natl Acad. Sci. USA, 91, 4955-4959. MEDLINE Abstract

15. Heilek,G.M., Marusak,R., Meares,C.F. and Noller,H.F. (1995)Proc. Natl Acad. Sci. USA, 92, 1113-1116. MEDLINE Abstract

16. Heilek,G.M. and Noller,H.F. (1996) Science, 272, 1659-1662. MEDLINE Abstract

17. Huq,I. and Rana,T.M. (1997) Biochemistry, 36, 12592-12599. MEDLINE Abstract

18. Jones,K.A. and Peterlin,B.M. (1994) Annu. Rev. Biochem., 63, 717-743. MEDLINE Abstract

19. Jeang,K.-T., Berkhout,B. and Dropulic,B. (1993) J. Biol. Chem., 268, 24940-24949. MEDLINE Abstract

20. Gaynor,R. (1992) AIDS, 6, 347-363. MEDLINE Abstract

21. Fisher,A.G., Feinberg,M.B., Josephs,S.F., Harper,M.E., Marselle,L.M., Reyes,G., Gonda,M.A., Aldovini,A., Debouck,C., Gallo,R.C. and Wong-Staal,F. (1986) Nature, 320, 367-371. MEDLINE Abstract

22. Dayton,A.I., Sodroski,J.G., Rosen,C.A., Goh,W.C. and Haseltine,W.A. (1986) Cell, 44, 941-947. MEDLINE Abstract

23. Cullen,B.R. (1992) Microbiol. Rev., 56, 375-394. MEDLINE Abstract

24. Berkhout,B., Silverman,R.H. and Jeang,K.T. (1989) Cell, 59, 273-282. MEDLINE Abstract

25. Cullen,B.R. (1986) Cell, 46, 973-982. MEDLINE Abstract

26. Peterlin,B.M., Luciw,P.A., Barr,P.J. and Walker,M.D. (1986) Proc. Natl Acad. Sci. USA, 83, 9734-9738. MEDLINE Abstract

27. Muesing,M.A., Smith,D.H. and Capon,D.A. (1987) Cell, 48, 691-701. MEDLINE Abstract

28. Rice,A.P. and Mathews,M.B. (1988) Nature, 332, 551-553. MEDLINE Abstract

29. Laspia,M.F., Rice,A.P. and Mathews,M.B. (1989) Cell, 59, 283-292. MEDLINE Abstract

30. Marciniak,R.A. and Sharp,P.A. (1991) EMBO J., 10, 4189-4196. MEDLINE Abstract

31. Kao,S.-Y., Calman,A.F., Luciw,P.A. and Peterlin,B.M. (1987) Nature, 330, 489-493. MEDLINE Abstract

32. Zhou,Q. and Sharp,P.A. (1995) EMBO J., 14, 321-328. MEDLINE Abstract

33. Graeble,M.A., Churcher,M.J., Lowe,A.D., Gait,M.J. and Karn,J. (1993) Proc. Natl Acad. Sci. USA, 90, 6184-6188. MEDLINE Abstract

34. Churcher,M.J., Lowe,A.D., Gait,M.J. and Karn,J. (1995) Proc. Natl Acad. Sci. USA, 92, 2408-2412. MEDLINE Abstract

35. Rosen,C.A., Sodroski,J.G. and Haseltine,W.A. (1985) Cell, 41, 813-823. MEDLINE Abstract

36. Jakobovits,A., Smith,D.H., Jakobovits,E.B. and Capon,D.J. (1988) Mol. Cell. Biol., 8, 2555-2561. MEDLINE Abstract

37. Selby,M.J., Bain,E.S., Luciw,P.A. and Peterlin,B.M. (1989) Genes Dev., 3, 547-558. MEDLINE Abstract

38. Garcia,J.A., Harrich,D., Soultanakis,E., Wu,F., Zmitsuyasu,R. and Gaynor,R.B. (1989) EMBO J., 8, 765-778. MEDLINE Abstract

39. 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) Proc. Natl Acad. Sci. USA, 86, 6925-6929. MEDLINE Abstract

40. Dingwall,C., Ernberg,I., Gait,M.J., Green,S.M., Heaphy,S., Karn,J., Lowe,A.D., Singh,M. and Skinner,M.A. (1990) EMBO J., 9, 4145-4153. MEDLINE Abstract

41. Feng,S. and Holland,E.C. (1988) Nature, 334, 165-167. MEDLINE Abstract

42. Berkhout,B. and Jeang,K.-T. (1989) J. Virol., 63, 5501-5504. MEDLINE Abstract

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) Proc. Natl Acad. Sci. USA, 87, 8985-8989. MEDLINE Abstract

44. Sumner-Smith,M., Roy,S., Barnett,R., Reid,L.S., Kuperman,R., Delling,U. and Sonenberg,N. (1991) J. Virol., 65, 5196-5202. MEDLINE Abstract

45. Han,H. and Dervan,P.B. (1994) Nucleic Acids Res., 22, 2837-2844. MEDLINE Abstract

46. Shah,K., Neenhold,H., Wang,Z. and Rana,T.M. (1996) Bioconjugate Chem., 7, 283-289.

47. Milligan,J.F., Groebe,D.R., Witherell,G.W. and Uhlenbeck,O.C. (1987) Nucleic Acids Res., 15, 8783-8798. MEDLINE Abstract

48. Wang,Z. and Rana,T.M. (1996) Biochemistry, 35, 6491-6499. MEDLINE Abstract

49. Neenhold,H.R. and Rana,T.M. (1995) Biochemistry, 34, 6303-6309. MEDLINE Abstract

50. Meares,C.F., McCall,M.J., Reardan,D.T., Goodwin,D.A., Diaminti,C.I. and McTigue,M. (1984) Anal. Biochem., 142, 68-78. MEDLINE Abstract

51. Fried,M. and Crothers,D.M. (1981) Nucleic Acids Res., 9, 6505-6525. MEDLINE Abstract

52. Calnan,B.J., Biancalana,S., Hudson,D. and Frankel,A.D. (1991) Genes Dev., 5, 201-210. MEDLINE Abstract

53. 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) J. Mol. Biol., 230, 90-110. MEDLINE Abstract

54. Delling,U., Reid,L.S., Barnett,R.W., Ma,M.Y.-X., Climie,S.,Summer-Smith,M. and Sonenberg,N. (1992) J. Virol., 66, 3018-3025. MEDLINE Abstract

55. Dreyer,G.B. and Dervan,P.B. (1985) Proc. Natl Acad. Sci. USA, 82, 968-972. MEDLINE Abstract

56. Balasubramanian,B., Pogozelski,W.K. and Tullius,T.D. (1998) Proc. Natl Acad. Sci. USA, 95, 9738-9743. MEDLINE Abstract

57. Aboul-ela,F., Karn,J. and Varani,G. (1996) Nucleic Acids Res., 24, 3974-3981. MEDLINE Abstract

58. Jaeger,J.A. and Tinoco,I.,Jr (1993) Biochemistry, 32, 12522-12530. MEDLINE Abstract

59. Weeks,K.M. and Crothers,D.M. (1991) Cell, 66, 577-588. MEDLINE Abstract

60. Puglisi,J.D., Tan,R., Calnan,B.J., Frankel,A.D. and Williamson,J.R. (1992) Science, 257, 76-80. MEDLINE Abstract

61. Moore,M.J. and Sharp,P.A. (1992) Science, 256, 992-997. MEDLINE Abstract

62. Aboul-ela,F., Karn,J. and Varani,G. (1995) J. Mol. Biol., 253, 313-332. MEDLINE Abstract

*To whom correspondence should be addressed. Tel: +1 732 235 4082; Fax:+1 732 235 3235; Email: rana@umdnj.edu

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