Solution conformation of an RNA hairpin loop.

The hairpin conformation adopted by the RNA sequence 5'GCGAUUUCUGACCGCC3' has been studied by one- and two-dimensional NMR spectroscopy. Exchangeable imino spectra in 60 mM Na+ indicate that the hairpin has a stem of six base pairs (indicated by boldface type) and a loop of three nucleotides. NOESY spectra of nonexchangeable protons confirm the formation of the stem region. The duplex has an A-conformation and contains an A.C apposition; a G.U base pair closes the loop region. The stem nucleotides have C3'-endo sugar conformations, as expected of an A-form duplex, whereas the three loop nucleotides adopt C2'-endo sugar puckers. Stacking within the loop, C8 upon the sugar of U7, stabilizes the structure. The pH dependence of both the exchangeable and nonexchangeable NMR spectra is consistent with the formation of an A+.C base pair, protonated at the N1 position of adenine. The stability of the hairpin was probed by using absorbance melting curves. The hairpin structure with the A+.C base pair is about +2 kcal/mol less stable in free energy at 37 degrees C than the hairpin formed with an A.U pair replacing the A+.C pair.     

Structural equilibria in RNA as revealed by 19F NMR

We have incorporated 5-fluorouridine into several sites within a 19-mer RNA modelled on the translational operator of the MS2 bacteriophage. The 19F NMR spectra demonstrate the different chemical shifts of helical and loop fluorouridines of the hairpin secondary structure. Addition of salt gives rise to a species in which the loop fluorouridine gains the chemical shift of its helical counterparts, due to the formation of the alternative bi-molecular duplex form. This is supported by UV thermal melting behaviour which becomes highly dependent on the RNA concentration. Distinct 19F NMR signals for duplex and hairpin forms allow the duplex-hairpin equilibrium constant to be determined under a range of conditions, enabling thermodynamic characterisation and its salt dependence to be determined. Mg2+ also promotes duplex formation, but more strongly than Na+, such that at 25 degrees C, 10 mM MgCl2 has a comparable duplex-promoting effect to 300 mM NaCl. A similar effect is observed with Sr2+, but not Ca2+ or Ba2+. Additional hairpin species are observed in the presence of Na+ as well as Mg2+, Ca2+, Sr2+ and Ba2+ ions. The overall, ensemble average, hairpin conformation is therefore salt-dependent. Electrostatic considerations are thus involved in the balance between different hairpin conformers as well as the duplex-hairpin equilibrium. The data presented here demonstrate that 19F NMR is a powerful tool for the study of conformational heterogeneity in RNA, which is particularly important for probing the effects of metal ions on RNA structure. The thermodynamic characterisation of duplex-hairpin equilibria will also be valuable in the development of theoretical models of nucleic acid structure.     

Structural characterization of a six-nucleotide RNA hairpin loop found in Escherichia coli, r(UUAAGU)

The binding region of the Escherichia coli S2 ribosomal protein contains a conserved UUAAGU hairpin loop. The structure of the hairpin formed by the oligomer r(GCGU4U5A6A7G8U9CGCA), which has an r(UUAAGU) hairpin loop, was determined by NMR and molecular modeling techniques as part of a study aimed at characterizing the structure and thermodynamics of RNA hairpin loops. Thermodynamic data obtained from melting curves for this RNA oligomer show that it forms a hairpin in solution with the following parameters: DeltaH degrees = -42.8 +/- 2.2 kcal/mol, DeltaS degrees = -127.6 +/- 6.5 eu, and DeltaG degrees (37) = -3.3 +/- 0.2 kcal/mol. Two-dimensional NOESY WATERGATE spectra show an NOE between U imino protons, which suggests that U4 and U9 form a hydrogen bonded U.U pair. The U5(H2') proton shows NOEs to both the A6(H8) proton and the A7(H8) proton, which is consistent with formation of a "U" turn between nucleotides U5 and A6. An NOE between the A7(H2) proton and the U9(H4') proton shows the proximity of the A7 base to the U9 sugar, which is consistent with the structure determined for the six-nucleotide loop. In addition to having a hydrogen-bonded U.U pair as the first mismatch and a U turn, the r(UUAAGU) loop has the G8 base protruding into the solvent. The solution structure of the r(UUAAGU) loop is essentially identical to the structure of an identical loop found in the crystal structure of the 30S ribosomal subunit where the guanine in the loop is involved in tertiary interactions with RNA bases from adjacent regions [Wimberly, B. T., Brodersen, D. E., Clemons, W. M., Morgan-Warren, R. J., Carter, A. P., Vonrhein, C., Hartsch, T., and Ramakrishnan, V. (2000) Nature 407, 327-339]. The similarity of the solution and solid-state structures of this hairpin loop suggests that formation of this hairpin may facilitate folding of 16S RNA.     

Structure of a small RNA hairpin.

The hairpin stem-loop form of the RNA oligonucleotide rCGC(UUU)GCG has been studied by NMR spectroscopy. In 10 mM phosphate buffer this RNA molecule forms a unimolecular hairpin with a stem of three base pairs and a loop of three uridines, as judged by both NMR and UV absorbance melting behavior. Distance and torsion angle restraints were determined using homonuclear proton-proton and heteronuclear proton-phosphorus 2-D NMR. These values were used in restrained molecular dynamics to determine the structure of the hairpin. The stem has characteristics of A-form geometry, although distortion from A-form occurs in the 3'-side of the stem, presumably to aid in accommodating the small loop. The loop nucleotides adopt C2'-endo conformations. NOE's strongly suggest stacking of the uracils with the stem, especially the first uracil on the 5'-side of the loop. The reversal of the chain direction in the loop seems to occur between U5 and U6. Loop structures produced by molecular dynamics simulations had a wide range of conformations and did not show stacking of the uracils. A flexible loop with significant dynamics is consistent with all the data.     

Interactions of hairpin oligo-2'-O-methylribonucleotides containing methylphosphonate linkages with HIV TAR RNA

Methylphosphonate-modified oligo-2'-O-methylribonucleotides 15-20 nucleotides (nt) in length were prepared whose sequences are complementary to the 5' and 3' sides of the upper hairpin of HIV trans-acting response element (TAR) RNA. These anti-TAR oligonucleotides (ODNs) form stable hairpins whose melting temperatures (Tm) range from 55 degrees C to 80 degrees C. Despite their rather high thermal stabilities, the hairpin oligo-2'-O-methylribonucleotides formed very stable complexes with TAR RNA, with dissociation constants in the nanomolar concentration range at 37 degrees C. The affinities of the hairpin oligomers for TAR RNA were influenced by the positions of the methylphosphonate linkages. The binding affinity was reduced approximately 17-fold by the presence of two methylphosphonate linkages in the TAR loop complementary region (TLCR) of the oligomer, whereas methylphosphonate linkages outside this region increased binding affinity approximately 3-fold. The configurations of the methylphosphonate linkages in the TLCR also affected binding affinity, with the RpRp isomer showing significantly higher binding than the SpSp isomer. In addition to serving as probes of the interactions between the oligomer and TAR RNA, the presence of the methylphosphonate linkages in combination with the hairpin structure increases the resistance of these oligomers to degradation by exonucleases found in mammalian serum. The combination of high binding affinity and nuclease resistance of the hairpin ODNs containing methylphosphonate linkages suggests their potential utility as antisense compounds.     

Structure of a single-stranded DNA target as a factor influencing the effectiveness of its modification with a complementary reagent]

Site directed alkylation of three oligonucleotide targets: 41-mer (hairpin structure), 22-mer (loop part of this hairpin) and 10-mer (part of the loop) with 5'-p-(N-2-chloroethyl-N-methylamino)benzylamides of oligonucleotides complementary to the loop region was studied. Thermodynamic parameters of the interaction were estimated using the dependence of the limit modification extent on the reagent concentration at different temperatures. The stability of the complex increases much in the set: 302-mer carrying the above hairpin, 41-mer, 22-mer; data on 22-mer and 10-mer being almost identical. This indicates significant influence of the loop supporting structure on the interaction with antisense reagents.     

The dimer initiation site hairpin mediates dimerization of the human immunodeficiency virus, type 2 RNA genome

The untranslated leader of retroviral RNA genomes encodes multiple structural signals that are critical for virus replication. In the human immunodeficiency virus, type 1 (HIV-1) leader, a hairpin structure with a palindrome-containing loop is termed the dimer initiation site (DIS), because it triggers in vitro RNA dimerization through base pairing of the loop-exposed palindromes (kissing loops). Controversy remains regarding the region responsible for HIV-2 RNA dimerization. Different studies have suggested the involvement of the transactivation region, the primer binding site, and a hairpin structure that is the equivalent of the HIV-1 DIS hairpin. We have performed a detailed mutational analysis of the HIV-2 leader RNA, and we also used antisense oligonucleotides to probe the regions involved in dimerization. Our results unequivocally demonstrate that the DIS hairpin is the main determinant for HIV-2 RNA dimerization. The 6-mer palindrome sequence in the DIS loop is essential for dimer formation. Although the sequence can be replaced by other 6-mer palindromes, motifs that form more than two A/U base pairs do not dimerize efficiently. The inability to form stable kissing-loop complexes precludes formation of dimers with more extended base pairing. Structure probing of the DIS hairpin in the context of the complete HIV-2 leader RNA suggests a 5-base pair elongation of the DIS stem as it is proposed in current RNA secondary structure models. This structure is supported by phylogenetic analysis of leader RNA sequences from different viral isolates, indicating that RNA genome dimerization occurs by a similar mechanism for all members of the human and simian immunodeficiency viruses.     

Hairpin Loop Structure in the 3′ Arm of the Influenza A Virus Virion RNA Promoter Is Required for Endonuclease Activity

Previous studies have shown that the 5' arm of the influenza A virus virion RNA promoter requires a hairpin loop structure for efficient endonuclease activity of influenza virus RNA polymerase, an activity that is required for the cap-snatching activity of primers from host pre-mRNA. Here we examine whether a hairpin loop is also required in the 3' arm of the viral RNA promoter. We study point mutations at each nucleotide position (1 to 12) within the 3' arm of the promoter as well as complementary "rescue" mutations which restored base pairing in the stem of a potential hairpin loop. Our results suggest that endonuclease activity is absolutely dependent on the presence of a 3' hairpin loop structure. This is the first direct evidence for RNA secondary structure within the 3' arm being required for a specific stage, i.e., endonuclease cleavage, in the influenza virus replicative cycle.     

Deletion of a single hydrogen bonding atom from the MS2 RNA operator leads to dramatic rearrangements at the RNA-coat protein interface

The MS2 coat protein binds specifically to an RNA hairpin formed within the viral genome. By soaking different RNA fragments into crystals of MS2 coat protein capsids it is possible to determine the X-ray structure of the RNA–protein complexes formed. Here we present the structure to 2.85 Å resolution of a complex between a chemically modified RNA hairpin variant and the MS2 coat protein. This RNA variant has a substitution at the –5 base position, which has been shown previously to be pyrimidine-specific and is a uracil in the wild-type RNA. The modified RNA hairpin contains a pyridin-4-one base (4one) at this position that lacks the exocyclic 2-oxygen eliminating the possibility of forming a hydrogen bond to asparagine A87 in the protein. The 4one complex structure shows an unprecedented major conformational change in the loop region of the RNA, whereas there is almost no change in the conformation of the protein.     

Structure of 2',5'-linked tetraribonucleotide loops: a novel RNA motif

We report on the three dimensional structure of an RNA hairpin containing a 2',5'-linked tetraribonucleotide loop, namely, 5'-rGGAC(UUCG)GUCC-3' (where UUCG = U(2'p5')U(2'p5')C(2'p5')G(2'p5')). We show that the 2',5'-linked RNA loop adopts a conformation that is quite different from that previously observed for the native 3',5'-linked RNA loop. The 2',5'-RNA loop is stabilized by (a) U:G wobble base pairing, with both bases in the anti conformation, (b) extensive base stacking, and (c) sugar-base contacts, all of which contribute to the extra stability of this hairpin structure.     

Substrate shape specificity of E coli RNase P ribozyme is dependent on the concentration of magnesium ion

The bacterial RNase P ribozyme can accept a hairpin RNA with CCA-3' tag sequence as well as a cloverleaf pre-tRNA as substrate in vitro, but the details are not known. By switching tRNA structure using an antisense guide DNA technique, we examined the Escherichia coli RNase P ribozyme specificity for substrate RNA of a given shape. Analysis of the RNase P reaction with various concentrations of magnesium ion revealed that the ribozyme cleaved only the cloverleaf RNA at below 10 mM magnesium ion. At 10 mM magnesium ion or more, the ribozyme also cleaved a hairpin RNA with a CCA-3' tag sequence. At above 20 mM magnesium ion, cleavage site wobbling by the enzyme in tRNA-derived hairpin occurred, and the substrate specificity of the enzyme became broader. Additional studies using another hairpin substrate demonstrated the same tendency. Our data strongly suggest that raising the concentration of metal ion induces a conformational change in the RNA enzyme.     

The 3'' terminus of 16S rRNA: secondary structure and interaction with ribosomal protein S1.

We report studies of the secondary structure and S1 ribosomal protein binding properties of the colicin fragment, containing 49 residues from the 3' terminus of E. coli 16S rRNA. Temperature jump relaxation kinetic measurements reveal two helices in the structure. One of these, melting at 81 degrees C in 5 mM Mg2+, is associated with the 9-base pair hairpin helix predicted by the nucleotide sequence. The other melting transition, at 21 degrees C in 5 mM Mg2+, is assigned to a 4-base pair helix which constrains the pyrimidine tract of the colicin fragment into a bulge loop. S1 protein forms a strong 1:1 complex with the colicin fragment, with an association constant of 5 x 10(6) M-1 in 5 mM Mg2+. More protein molecules are bound, but with weaker affinity, when the S1 concentration is increased. S1 binding causes melting of the colicin fragment secondary structure, as inferred from the observed absorbance increase. The S1 binding site on the colicin fragment has been localized in the region of the bulge loop, since the melting transition corresponding to the 4-base pair helix is lost in the complex. We discuss current models for the role of S1 protein in polypeptide chain initiation in light of these and previous results.     

An NMR Study of the Polymorphous Behavior of the Mismatched DNA Octamer d(m5C-G-m5C-G-T-G-m5C-G) in Solution. The B,Z, and Hairpin Forms

Abstract

The polymorphism exhibited by the mismatched octamer d(m ⁵C-G-m⁵C-G-T-G-m⁵C-G), as a function of the temperature, DNA concentration and ionic strength, was investigated by means of NMR spectroscopy.

It is shown that this partly self-complementary DNA fragment, under conditions of low DNA concentration (0.4 mM) and low ionic strength, exclusively prefers to adopt a monomeric hairpin form, which consists of a stem of three Watson-Crick-type base pairs and a loop of only two residues. This in striking contrast with earlier intimations in literature, which postulated that in oligonucleotides loop formations containing only two residues are sterically impossible. Moreover, the hairpin form displays an unusual stability in comparison with previously reported hairpins. AT_m of 332 K and a ΔH° of—130 kj · mol^?1 were calculated for the hairpin to random coil transition.

At high DNA concentration (8 mM)and/or upon the addition of sodium chloride the hairpin form occurs in slow exchange with a B-DNA dimer structure (approximately 20% at 270 K, no added salt), which comprises two central GxT-mismatched base pairs with the bases as major tautomers.

At higher ionic strength (> 100 mM NaCI), or upon the addition of methanol, a third species appears, which is in slow exchange with both the B dimer and the hairpin form. This third species could be identified with a Z DNA form, comprising two GxT mismatches with the bases as major tautomers, with the guanine bases syn and the cytosine and thymine bases anti.     

Base and sugar requirements for RNA cleavage of essential nucleoside residues in internal loop B of the hairpin ribozyme: implications for secondary structure.

The hairpin ribozyme is a small self-cleaving RNA that can be engineered for RNA cleavage in trans and has potential as a therapeutic agent. We have used a chemical synthesis approach to study the requirements of hairpin RNA cleavage for sugar and base moieties in residues of internal loop B, an essential region in one of the two ribozyme domains. Individual nucleosides were substituted by either a 2'-deoxy-nucleoside, an abasic residue, or a C3-spacer (propyl linker) and the abilities of the modified ribozymes to cleave an RNA substrate were studied in comparison with the wild-type ribozyme. From these results, together with previous studies, we propose a new model for the potential secondary structure of internal loop B of the hairpin ribozyme.     

Mutational analysis of a conserved tetraloop in the 5' untranslated region of hepatitis C virus identifies a novel RNA element essential for the internal ribosome entry site function.

The 5' untranslated region of hepatitis C virus RNA forms an extensive secondary structure including several hairpin motifs and mediates translation initiation by an internal ribosome entry site-dependent pathway. We report, here, an extensive mutagenesis analysis of a highly conserved tetraloop in the 5' untranslated region of hepatitis C virus, namely hairpin IIIe (295'-GAUA-298'). Our results demonstrate that hairpin IIIe is essential for the internal ribosome entry site function. Moreover, they indicate the importance of the primary structure of this motif because mutations in all four nucleotides of the loop caused a severe loss of internal ribosome entry site activity. These data represent the first experimental evidence for the functional significance of tetraloops in internal ribosome entry site-driven translation of hepatitis C virus.     

Mg2+ Dependence of the Structure and Thermodynamics of Wheat Germ and Lupin Seeds 5S rRNA

Abstract

The formation and stability of structural elements in two 5S rRNA molecules from wheat germ (WG) and lupin seeds (LS) as a function of Mg²⁺ concentration in solution was determined using the adiabatic differential scanning microcalorimetry (DSC). The experimentally determined thermodynamic parameters are compared with calculations using thermodynamic databases used for prediction of RNA structure. The 5S rRNA molecules which show minor differences in the nucleotide sequence display very different thermal unfolding profiles (DSC profiles). Numerical deconvolution of DSC profiles provided information about structural transformations that take place in both 5S rRNA molecules. A comparative analysis of DSC data and the theoretical thermodynamic models of the structure was used to establish a relationship between the constituting transitions found in the melting profiles and the unfolding of structural domains of the 5S rRNA and stability of its particular helical elements.

Increased concentration of Mg²⁺ ions induces additional internal interactions stabilising 5S rRNA structures found at low Na⁺ concentrations. Observed conformational transitions suggest a structural model in which the extension of helical region E dominates over the postulated tertiary interaction between hairpin loops. We propose that helix E is stabilised by a sequence of non-standard pairings extending this helix by the formation of tetra loop e and an almost total reduction of loop d between helices E and D. Two hairpin structures in both 5S rRNA molecules: the extended C-C' and the extended E-E'-E” hairpins appear as the most stable elements of the structure. The cooperativity of the unfolding of helixes in these 5S rRNA molecules changes already at 2 mM Mg²⁺.