Major groove opening at the HIV-1 Tat binding site of TAR RNA evidenced by a rhodium probe

Transactivation of human immunodeficiency virus (HIV) gene expression requires the interaction of Tat protein with the trans-activation responsive region (TAR) RNA, a 59-base stem-loop structure located at the 5'-end of all mRNAs. The TAR RNA contains a six-nucleotide loop and a three-nucleotide pyrimidine bulge which separates two helical stem regions. The trinucleotide bulge is essential for high affinity and specific binding of the Tat protein. Recently, a rhodium complex, Rh(phen)2phi3+, was discovered which promotes RNA cleavage in the open major groove and triply bonded bases [Chow, C. S., et al. (1992) Biochemistry 31, 972-982]. This metal complex does not bind double-helical RNA or unstructured single-stranded regions of RNA. Instead, sites of tertiary interaction which are open in the major groove and accessible to stacking are targeted by the complex through photoactivated cleavage. We have used this rhodium probe to investigate the effect of bulge bases on the major groove opening in TAR RNA. 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. A strong cleavage at residues C39 and U40 was observed on the wild-type TAR RNA and in mutant TAR RNA containing two mismatch bases in the bulge. No cleavage at C39 and U40 was observed in a bulgeless and a one-base bulge TAR RNA. By varying the number of mismatch bases, we demonstrated that the trinuclear bulge widens the major groove of TAR RNA to facilitate Tat binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

A 1.3-A resolution crystal structure of the HIV-1 trans-activation response region RNA stem reveals a metal ion-dependent bulge conformation

The crystal structure of an HIV-1 trans-activation response region (TAR) RNA fragment containing the binding site for the trans-activation protein Tat has been determined to 1.3-A resolution. In this crystal structure, the characteristic UCU bulge of TAR adopts a conformation that is stabilized by three divalent calcium ions and differs from those determined previously by solution NMR. One metal ion, crucial to the loop conformation, binds directly to three phosphates in the loop region. The structure emphasizes the influence of metal ion binding on RNA structure and, given the abundance of divalent metal ion in the cell, raises the question of whether metal ions play a role in the conformation of TAR RNA and the interaction of TAR with Tat and cyclin T in vivo.     

Rotational symmetry in ribonucleotide strand requirements for binding of HIV-1 Tat protein to TAR RNA

Transactivation of human immunodeficiency virus (HIV) gene expression requires binding of the viral Tat protein to a RNA hairpin-loop structure (TAR) which contains a two or three-nucleotide bulge. Tat binds in the vicinity of the bulge and the two adjacent duplex stems, recognising both specific sequence and structural features of TAR. Binding is mediated by an arginine-rich domain, placing Tat in the family of arginine-rich RNA binding proteins that includes other transactivators, virus capsid proteins and ribosome binding proteins. In order to determine what features of TAR allow Tat to bind efficiently to RNA but not DNA forms, we examined Tat binding to a series of RNA-DNA hybrids. We found that only one specific strand in each duplex stem region needs to be RNA, implying that interaction between Tat and a given stem may be solely or predominantly with one of the two strands. However, the essential strand is not the same one for each stem, suggesting a switch in the bound strand on opposing sides of the bulge.     

Mechanism of substrate recognition by the ribotoxin, alpha-sarcin

The substrate recognition and catalytic mechanisms of alpha-sarcin were explored with kinetic method by using synthetic 25-mer RNA mimicking the alpha-sarcin/ricin loop in 23S rRNA of E. coli ribosomes. The oligomer containing deoxy-G at the site of alpha-sarcin (G14) was a potent competitive inhibitor. The RNA having deoxy-G8 however, increases the Kcat value by about five times but without significant alteration on Km. Surprisingly, the deletion of G8 makes the oligomer become a strong noncompetitive inhibitor of the enzyme. These results suggested that there are at least two sites in the RNA substrate which are recognized by alpha-sarcin, one is the G8 bulge or at around its neighbor and the other is the GAGA in the sarcin/ricin loop of the rRNA.     

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 basic region

The binding site for tat protein on TAR RNA has been defined in quantitative terms using an extensive series of mutations. The relative dissociation constants for the mutant TAR RNAs were measured using a dual-label competition filter binding assay in which 35S-labelled wild-type TAR RNA (K1) was competed against 3H-labelled mutant TAR RNA (K2). The error in the self-competition experiment was usually less than 10% (e.g. K2/K1 = 1.07 +/- 0.05, n = 19) and the experimental data accurately matched theoretical curves calculated with fitted dissociation constants. Mutations in U23, a critical residue in the U-rich "bulge" sequence, or in either of the two base-pairs immediately above the "bulge", G26.C39 and A27.U38 reduced that affinity by 8- to 20-fold. Significant contributions to tat binding affinity were also made by the base-pairs located immediately below the bulge. For example, mutation of A22.U40 to U.A reduced tat affinity 5-fold, and mutation of G21.C41 to C.G reduced tat affinity 4-fold. The binding of a series of peptides spanning the basic "arginine-rich" sequence of tat was examined using both filter-binding and gel mobility shift assays. Each of the peptides showed significantly reduced affinities for wild-type TAR RNA compared to the tat protein. The ADP-2 (residues 43 to 72), ADP-3 (residues 48 to 72) and ADP-5 (residues 49 to 86) peptides were unable to discriminate between wild-type TAR RNA and TAR RNA mutants with the same fidelity as the tat protein. For example, these peptides showed no more than 3-fold reductions in affinity relative to wild-type TAR RNA for the U23-->C mutation in the bulge, or G26.G39-->C.G mutation in the stem of TAR RNA. By contrast, the ADP-I (residues 37 to 72), ADP-4 (residues 32 to 62) and ADP-6 (residues 32 to 72) peptides, which each carry amino acid residues from the "core" region of the tat protein have binding specificities that more closely resemble the protein. The ADP-4 and ADP-6 peptides showed between 4- and 7-fold reductions in affinity for the U23-->C or G26.C39-->C.G mutations. The ADP-1 peptide most closely resembles the protein in its binding specificity and showed 9-fold and 14-fold reductions in affinity for the two mutants, respectively. Chemical-modification interference assays using diethylpyrocarbonate (DEPC) and ethylnitrosourea (ENU) were also used to compare the binding properties of the tat protein and the tat-derived peptides.(ABSTRACT TRUNCATED AT 400 WORDS)     

Structure-activity correlation for an HDV ribozyme composed of three RNA strands

Hepatitis delta virus (HDV) RNA ribozyme system which consists of three RNA oligomer strands (substrate 8-mer; enzyme 16-mer plus 35-mer, Fig. 1) was designed. Effects of Mg2+ concentration on the pseudo first-order rate constant (kobs) of RNA cleavage reaction and on conformation of ribozyme complex were examined. The secondary structure of the complex was also analyzed by limited digestion with ribonucleases. The kobs and CD data were analyzed by curve-fitting analysis using equations derived for two-Mg2+ and three-Mg2+ ion binding models. The result revealed that a three-Mg2+ binding model can explain the Mg(2+)-concentration-dependent changes of both conformation and activity of the HDV ribozyme.     

Selective recognition and cleavage of RNA loop structures by Ni(II).Xaa-Gly-His metallopeptides

The recognition and cleavage of tRNAPhe and the TAR RNA of HIV-1 by metallopeptides of the general form Ni(II).Xaa-Gly-His (where Xaa is Gly, Lys, or Arg) were investigated. The results of RNA cleavage analyses suggest that KHSO5- or magnesium monoperoxyphthalate-activated metallopeptides (1) induce nucleobase damage which requires aniline acetate for complete RNA strand scission and (2) selectively target the loops of stem-loop structures of the above-named substrates. In targeting RNA loop regions, the metallopeptides may be sensitive to intraloop structural features, including the overall structural environment of the loop itself and possibly the presence of intraloop hydrogen bonding. Overall, these results suggest that the metallopeptides interact selectively within a loop, in a fashion reminiscent of many RNA binding proteins, instead of targeting RNA single-stranded character alone. These observations further suggest a possible metallopeptide-based strategy for the molecular recognition of native RNA structures and insight with regard to the general features available for ligand binding site discrimination.     

TAR-RNA binding by HIV-1 Tat protein is selectively inhibited by its L-enantiomer

An oligoribonucleotide, corresponding to the Tat-interactive top half of the HIV-1 TAR RNA stem-loop, was synthesized in both the natural D- and the enantiomeric L-configurations. The affinity of Tat for the two RNAs, assessed by competition binding experiments, was found to be identical and is reduced 10-fold for both, upon replacement of the critical bulge residue U23 with cytidine. It is suggested that this interaction of the flexible Tat protein depends strongly upon the tertiary structure of a binding pocket within TAR, but not upon its handedness, and may be described by a 'hand-in-mitten' model.     

Metal ion-dependent hydrolysis of RNA phosphodiester bonds within hairpin loops. A comparative kinetic study on chimeric ribo/2'-O-methylribo oligonucleotides

Several chimeric ribo/2'- O -methylribo oligonucleotides were synthesized and their hydrolytic cleavage studied in the presence of Mg2+, Zn2+, Pb2+and the 1,4,9-triaza-cyclododecane chelate of Zn2+(Zn2+[12]aneN3) to evaluate the importance of RNA secondary structure as a factor determining the reactivity of phosphodiester bonds. In all the cases studied, a phosphodiester bond within a 4-7 nt loop was hydrolytically more stable than a similar bond within a linear single strand, but markedly less stable than that in a double helix. With Zn2+and Zn2+[12]aneN3, the hydrolytic stability of a phosphodiester bond within a hairpin loop gradually decreased on increasing the distance from the stem. A similar but less systematic trend was observed with Pb2+. Zn2+- and Pb2+-promoted cleavage was observed to be considerably more sensitive to the secondary structure of the chain than that induced by Zn2+[12]aneN3. This difference in behaviour may be attributed to bidentate binding of uncomplexed aquo ions to two different phosphodiester bonds. Mg2+was observed to be catalytically virtually inactive compared with the other cleaving agents studied.     

Relatedness of an RNA-binding motif in human immunodeficiency virus type 1 TAR RNA-binding protein TRBP to human P1/dsI kinase and Drosophila staufen

TRBP is a human cellular protein that binds the human immunodeficiency virus type 1 TAR RNA. Here, we show that the intact presence of amino acids 247 to 267 in TRBP correlates with its ability to bind RNA. This region contains a lysine- and arginine-rich motif, KKLAKRNAAAKMLLRVHTVPLDAR. A 24-amino-acid synthetic peptide (TR1) of this sequence bound TAR RNA with affinities similar to that of the entire TRBP, thus suggesting that this short motif contains a sufficient RNA-binding activity. Using RNA probe-shift analysis, we determined that TR1 does not bind all double-stranded RNAs but prefers TAR and other double-stranded RNAs with G+C-rich characteristics. Immunoprecipitation of TRBP from human immunodeficiency virus type 1-infected T lymphocytes recovered TAR RNA. This is consistent with a TRBP-TAR ribonucleoprotein during viral infection. Computer alignment revealed that TR1 is highly homologous to the RNA-binding domain of human P1/dsI protein kinase and two regions within Drosophila Staufen. We suggest that these proteins are related by virtue of sharing a common RNA-binding moiety.     

The 5' stem-loop from human U4 snRNA adopts a novel conformation stabilized by G-C and G-U base pairs

1H NMR and molecular modeling studies of the 5' stem-loop from human U4 snRNA were undertaken to determine the conformation of this stem-loop that is essential for spliceosome formation and pre-mRNA splicing. Sixteen of the 35 nucleotides of this stem-loop are in the loop region and inspection of the loop sequence revealed no decomposition into elements of secondary structure commonly found in other RNA stem-loops. An analysis of possible base pairing interactions for this stem-loop using the methods of Zuker revealed the lowest energy secondary structure for the 16 nucleotide loop consisted of four base pairs at the base of a non-canonical tetraloop (UUUA). This shorter stem-loop was joined to the nine base pair stem by two A residues on the 5' side and a single bulged A on the 3' side. Both stems also had bulged A residues. 1H NMR experiments performed on solutions of the 35 mer stem-loop, the stem region, and the loop region confirmed the 35 mer adopted this secondary structure in solution. A 3D molecular model of this structure consistent with the NMR data was generated to assist in visualization of this novel structure.     

Central sleep apnoea and heart failure (part II)

The three-dimensional (3-D) structure of a RNA pseudoknot that causes the efficient ribosomal frameshifting in the gag-pro region of mouse mammary tumor virus (MMTV) has been determined recently by nuclear magnetic resonance (NMR) studies. But since the structure refinement in the studies did not use metal ions and waters, it is not clear how metal ions participate in the stabilization of the pseudoknot, and what kind of ion-RNA interactions dominate in the tertiary contacts for the RNA pseudoknotting. Based on the reported structure data of the pseudoknot VPK of MMTV, we gradually refined the structure by restrained molecular dynamics (MD) using NMR distance restraints. Restrained MD simulation of the RNA pseudoknot was performed with sodium ions and water molecules. Our results are in good agreement with known NMR data and delineate the importance of the metal ion coordination in the stability of the pseudoknot. In the non-coaxially stacking pseudoknot, stem 1 (S1), stem 2 (S2), and the intervening A14 involves unconventional stacking of base pairs coordinated by Na+ and/or bridging water molecules. A6 and G7 of loop L1 make a perfect base stacking in the major groove and are further stabilized by coordinated Na+ ions and water molecules. The first 4-nucleotide (nt) ACUC of loop L2 form a sharp turn and the following 4-nt AAAA cross the minor groove of S1 and are steadied by interactions with the nucleotides of S , bridging water molecules and coordinated Na+ ions. Our studies suggest that the metal ion plays a crucial role in the RNA pseudoknotting of VPK. In the stacking interior of S1 and S2, the Na+ ion is positioned in the major groove and interacts directly with the carbonyl group O6 of G28 and carbonyl group O4 of U13 in the wobble base pair U13:G28. The ion-RNA interactions in MMTV VPK not only stabilize the RNA pseudoknot but also modify the electrostatic properties of the nucleotides at the critical parts of the pseudoknot VPK.     

RNA bulge entropies in the unbound state correlate with peptide binding strengths for HIV-1 and BIV TAR RNA because of improved conformational access

For the binding of peptides to wild-type HIV-1 and BIV TAR RNA and to mutants with bulges of various sizes, changes in the DeltaDelta G values of binding were determined from experimental K d values. The corresponding entropies of these bulges are estimated by enumerating all possible RNA bulge conformations on a lattice and then applying the Boltzmann relationship. Independent calculations of entropies from fluctuations are also carried out using the Gaussian network model (GNM) recently introduced for analyzing folded structures. Strong correlations are seen between the changes in free energy determined for binding and the two different unbound entropy calculations. The fact that the calculated entropy increase with larger bulge size is correlated with the enhanced experimental binding free energy is unusual. This system exhibits a dependence on the entropy of the unbound form that is opposite to usual binding models. Instead of a large initial entropy being unfavorable since it would be reduced upon binding, here the larger entropies actually favor binding. Several interpretations are possible: (i) the higher conformational freedom implies a higher competence for binding with a minimal strain, by suitable selection amongst the set of already accessible conformations; (ii) larger bulge entropies enhance the probability of the specific favorable conformation of the bound state; (iii) the increased freedom of the larger bulges contri-butes more to the bound state than to the unbound state; (iv) indirectly the large entropy of the bound state might have an unfavorable effect on the solvent structure. Nonetheless, this unusual effect is interesting.