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.     

Non-canonical interactions in a kissing loop complex: the dimerization initiation site of HIV-1 genomic RNA

Retroviruses encapsidate two molecules of genomic RNA that are noncovalently linked close to their 5' ends in a region called the dimer linkage structure (DLS). The dimerization initiation site (DIS) of human immunodeficiency virus type 1 (HIV-1) constitutes the essential part of the DLS in vitro and is crucial for efficient HIV-1 replication in cell culture. We previously identified the DIS as a hairpin structure, located upstream of the major splice donor site, that contains in the loop a six-nucleotide self-complementary sequence preceded and followed by two and one purines, respectively. Two RNA monomers form a kissing loop complex via intermolecular interactions of the six nucleotide self-complementary sequence. Here, we introduced compensatory mutations in the self-complementary sequence and/or a mutation in the flanking purines. We determined the kinetics of dimerization, the thermal stabilities and the apparent equilibrium dissociation constants of wild-type and mutant dimers and used chemical probing to obtain structural information. Our results demonstrate the importance of the 5'-flanking purine and of the two central bases of the self-complementary sequence in the dimerization process. The experimental data are rationalized by triple interactions between these residues in the deep groove of the kissing helix and are incorporated into a three-dimensional model of the kissing loop dimer. In addition, chemical probing and molecular modeling favor the existence of a non-canonical interaction between the conserved adenine residues at the first and last positions in the DIS loop. Furthermore, we show that destabilization of the kissing loop complex at the DIS can be compensated by interactions involving sequences located downstream of the splice donor site of the HIV-1 genomic RNA.     

Inter-domain cross-linking and molecular modelling of the hairpin ribozyme

The hairpin ribozyme is a small catalytic RNA composed of two helical domains containing a small and a large internal loop and, thus, constitutes a valuable paradigm for the study of RNA structure and catalysis. We have carried out molecular modelling of the hairpin ribozyme to learn how the two domains (A and B) might fold and approach each other. To help distinguish alternative inter-domain orientations, we have chemically synthesized hairpin ribozymes containing 2'-2' disulphide linkages of known spacing (12 or 16 A) between defined ribose residues in the internal loop regions of each domain. The abilities of cross-linked ribozymes to carry out RNA cleavage under single turnover conditions were compared to the corresponding disulphide-reduced, untethered ribozymes. Ribozymes were classed in three categories according to whether their cleavage rates were marginally, moderately, or strongly affected by cross-linking. This rank order of activity guided the docking of the two domains in the molecular modelling process. The proposed three-dimensional model of the hairpin ribozyme incorporates three different crystallographically determined structural motifs: in domain A, the 5'-GAR-3'-motif of the hammerhead ribozyme, in domain B, the J4/5 motif of group I ribozymes, and connecting the two domains, a "ribose zipper", another group I ribozyme feature, formed between the hydroxyl groups of residues A10, G11 of domain A and C25, A24 of domain B. This latter feature might be key to the selection and precise orientation of the inter-domain docking necessary for the specific phosphodiester cleavage. The model provides an important basis for further studies of hairpin ribozyme structure and function.     

Molecular recognition in the FMN-RNA aptamer complex

We report on a combined NMR-molecular dynamics calculation approach that has solved the solution structure of the complex of flavin mononucleotide (FMN) bound to the conserved internal loop segment of a 35 nucleotide RNA aptamer identified through in vitro selection. The FMN-RNA aptamer complex exhibits exceptionally well-resolved NMR spectra that have been assigned following application of two, three and four-dimensional heteronuclear NMR techniques on samples containing uniformly 13C, 15N-labeled RNA aptamer in the complex. The assignments were aided by a new through-bond NMR technique for assignment of guanine imino and adenine amino protons in RNA loop segments. The conserved internal loop zippers up through the formation of base-pair mismatches and a base-triple on complex formation with the isoalloxazine ring of FMN intercalating into the helix between a G.G mismatch and a G.U.A base-triple. The recognition specificity is associated with hydrogen bonding of the uracil like edge of the isoalloxazine ring of FMN to the Hoogsteen edge of an adenine at the intercalation site. There is significant overlap between the intercalated isoalloxazine ring and its adjacent base-triple platform in the complex. The remaining conserved residues in the internal loop participate in two G.A mismatches in the complex. The zippered-up internal loop and flanking stem regions form a continuous helix with a regular sugar-phosphate backbone except at a non-conserved adenine, which loops out of the helix to facilitate base-triple formation. Our solution structure of the FMN-RNA aptamer complex is to our knowledge the first structure of an RNA aptamer complex and outlines folding principles that are common to other RNA internal and hairpin loops, and molecular recognition principles common to model self-replication systems in chemical biology.     

Hairpin-dimer equilibrium of a parallel-stranded DNA hairpin: formation of a four-stranded complex

The 24mer deoxyoligonucleotide 3'-d(T)10-5'-5'-d(C)4- d(A)10-3'(psC4) with an uncommon 5'-p-5'phosphodiester linkage was designed to enable the formation of a hairpin structure with unusual parallel-stranded stem. As reference hairpin structure with an antiparallel-stranded stem, the 24mer 5'-d(T)10-d(C)4-d(A)10-3'(apsC4) was chosen. The behaviour of these oligonucleotides at different temperatures, DNA and salt concentrations was characterised by a combination of UV melting, CD, CD melting, infrared and Raman spectroscopy, infrared melting and analytical ultracentrifugation. The parallel-stranded hairpin structure was found to be formed by psC4 only under conditions of low DNA concentration and low salt concentration. Increase of the NaCl concentration beyond the physiological level or high DNA concentration supports the formation of intermolecular multi-stranded structures. The experimental data are in agreement with a four-stranded complex formed by two molecules of psC4. The base pairing model of this asymmetric four-stranded complex is based on the pyrimidine motif of a triple helix with two bifurcated hydrogen bonds at the O4 of the thymine each directed towards one of the amino protons of both adenines. In contrast, the reference oligonucleotide apsC4 forms only an antiparallel-stranded hairpin under all experimental conditions.     

The structure of the isolated, central hairpin of the HDV antigenomic ribozyme: novel structural features and similarity of the loop in the ribozyme and free in solution

The structure of an RNA hairpin containing a seven-nucleotide loop that is present in the self-cleaving sequence of hepatitis delta virus antigenomic RNA was determined by high resolution NMR spectroscopy. The loop, which is composed of only one purine and six pyrimidines, has a suprisingly stable structure, mainly supported by sugar hydroxyl hydrogen bonds and base-base and base-phosphate stacking interactions. Compared with the structurally well-determined, seven-membered anticodon loop in tRNA, the sharp turn which affects the required 180 degrees change in direction of the sugar-phosphate backbone in the loop is shifted one nucleotide in the 3' direction. This change in direction can be characterized as a reversed U-turn. It is expected that the reversed U-turn may be found frequently in other molecules as well. There is evidence for a new non-Watson-Crick UC base pair formed between the first and the last residue in the loop, while most of the other bases in the loop are pointing outwards making them accessible to solvent. From chemical modification, mutational and photocrosslinking studies, a similar picture develops for the structure of the hairpin in the active ribozyme indicating that the loop structure in the isolated hairpin and in the ribozyme is very similar.     

Secondary structure dimorphism and interconversion between hairpin and duplex form of oligoribonucleotides

RNA hairpins can alternatively form a dimer with a bulged loop flanked by regularly base paired regions. [1H]NMR spectroscopy and native gel electrophoresis were used to study how the sequence of nucleotides in the loop of the hairpin affect the hairpin-duplex interconversion. As a model system, a hairpin containing 7 nucleotides in the loop and 5 base pairs in the stem was used. The loop size was gradually reduced from 7 to 4 nucleotides, yielding finally the stable UNCG tetraloop. Single nucleotide mutations were performed to investigate the influence of the self-complementarity of the loop sequence on the dimerization. The results demonstrate that (1) the initial fraction of hairpin is determined by concentration of the oligonucleotide, the annealing procedure, and the relative stability of the loop, (2) the degree of self-complementarity of the loop sequence of the hairpin governs the dimerization kinetics, and (3) oligonucleotides complementary to the loop sequence decrease the dimerization rate. We propose a secondary structure-based model for the dimerization reaction of RNA hairpins in which the formation of intermolecular base pairs between self-complementary nucleotides of the loops represents the nucleation step.     

A minor groove RNA triple helix within the catalytic core of a group I intron

Close packing of several double helical and single stranded RNA elements is required for the Tetrahymena group I ribozyme to achieve catalysis. The chemical basis of these packing interactions is largely unknown. Using nucleotide analog interference suppression (NAIS), we demonstrate that the P1 substrate helix and J8/7 single stranded segment form an extended minor groove triple helix within the catalytic core of the ribozyme. Because each triple in the complex is mediated by at least one 2'-OH group, this substrate recognition triplex is unique to RNA and is fundamentally different from major groove homopurine-homopyrimidine triplexes. We have incorporated these biochemical data into a structural model of the ribozyme core that explains how the J8/7 strand organizes several helices within this complex RNA tertiary structure.     

RNA-RNA interactions between oligonucleotide substrates for aminoacylation

RNA stem-loop microhelices with helix sequences based on tRNA acceptor stems can be charged with specific amino acids. Experiments were designed to test the possibility that microhelices could laterally associate through complementary loop sequences and thereby bring their attached aminoacyl groups close enough together to form a peptide bond. Computer simulations suggested that formation of such complexes would be sensitive to the number of loop nucleotides needed to span the grooves of the quasi-continuous helix of the intermolecular pseudoknot so formed. These predictions were conformed experimentally by observation of complex formation sensitivity to loop size. Complexes with optimized loop sizes had apparent bimolecular dissociation constants of approximately 100 nM with only three complementary base pairs between the respective loops. Single nucleotide substitutions that disrupted the predicted intermolecular loop-loop base-pairing abolished detectable association. Similarly, placing a gap between the short helix formed by loop-loop pairing and the adjacent acceptor stems also diminished complex formation. These experiments establish an experimental basis for microhelix association for peptide synthesis.     

Structure and activity of the hairpin ribozyme in its natural junction conformation: effect of metal ions

The natural form of the hairpin ribozyme consists of a four-way RNA junction of which the single-stranded loop-carrying helices are adjacent arms. The junction can be regarded as providing a framework for constructing the active ribozyme, and the rate of cleavage can be modulated by changing the conformation of the junction. We find that the junction-based form of the hairpin ribozyme is active in magnesium, calcium, or strontium ions, but not in manganese, cadmium, or sodium ions. Using fluorescence resonance energy transfer experiments, we have investigated the global structure of the ribozyme. The basic folding of the construct is based on pairwise helical stacking, so that the two loop-carrying arms are located on opposite stacked helical pairs. In the presence of magnesium, calcium, or strontium ions, the junction of the ribozyme undergoes a rotation into a distorted antiparallel geometry, creating close physical contact between the two loops. Manganese ions induce the same global folding, but no catalytic activity; this change in global conformation is therefore necessary but not sufficient for catalytic activity. Fitting the dependence of the conformation on ionic concentration to a two-state model suggests that cooperative binding of two ions is required to bring about the folding. However, further ion binding is required for cleavage activity. Cobalt hexammine ions also bring about global folding, while spermidine generates a more symmetrical form of the antiparallel structure. Cadmium ions generate a different folded form, interpreted in terms of close loop-loop association while the junction is unfolded. Sodium ions were unable to induce any folding of the ribozyme, which remained slightly parallel. These results are consistent with a folding process induced by the binding of two group IIA metal ions, distributed between the junction and the loop interface.     

Structure of neurotoxin B-IV from the marine worm Cerebratulus lacteus: a helical hairpin cross-linked by disulphide bonding

B-IV is a 55-residue, crustacean-selective, neurotoxin secreted by Cerebratulus lacteus, a large marine worm found along the northeastern coast of North America. The 3D structure of this molecule in aqueous solution has been determined by 1H NMR spectroscopy at 600 MHz. The molecule has a well-defined helical hairpin structure, with the branches of the hairpin linked by four disulphide bonds. The disulphide connectivities were established from the NMR data to be 1-8/2-7/3-6/4-5, which differed from those determined previously by chemical means, where 1-7 and 2-8 connectivities were found. Each branch of the hairpin is largely alpha-helical, with the helices in the N and C-terminal branches encompassing residues 11 to 23 and 34 to 49, respectively. The loop connecting the branches of the hairpin contains two inverse gamma-turns centred on residues 24 and 25, a type I beta-turn at residues 28 to 31 and a type II beta-turn at residues 30 to 33. Arg17, -25 and -34, which are important for activity, are all on the same face of the molecule, while Trp30, which is also important for activity, is on the opposite face. Structure comparisons show that the B-IV structure is quite similar to those of Rop (ColE1 repressor of primer) and the heat-stable enterotoxin B from Escherichia coli. These structural similarities are discussed in relation to possible mechanisms of action of B-IV.     

13C relaxation and dynamics of the purine bases in the iron responsive element RNA hairpin

The iron responsive element (IRE) RNA hairpin contains a conserved six-nucleotide loop. The NMR structure of this loop showed that the positions of four of its bases are not tightly constrained, while the remaining two are hydrogen-bonded [Laing, L. G., and Hall, K. B. (1996) Biochemistry 35, 13586]. To investigate the flexibility of the RNA in the loop and in the stem, 13C NMR relaxation methods have been used to describe the dynamics of the purine bases. IRE hairpins containing [13C]guanosine and [13C]adenosine are used in NMR experiments to measure T1, T1rho, and NOE values of the bases as a function of temperature (20-37 degreesC). Data are analyzed using the Lipari-Szabo model-free formalism [Lipari, G., and Szabo, A. (1982) J. Am. Chem. Soc. 104, 4546] to determine order parameters and time scales of the motion. Results indicate that the purine bases in the stem have order parameters that are independent of temperature, although they show evidence of both fast (6-40 ps) motions and slower motions at 37 degreesC. The three purines in the loop exhibit increasingly complex motions with long (nanoseconds) correlation times as the temperature increases, suggesting that the loop structure has become disordered.     

NMR structure and dynamics of an RNA motif common to the spliceosome branch-point helix and the RNA-binding site for phage GA coat protein

The RNA molecules that make up the spliceosome branch-point helix and the binding site for phage GA coat protein share a secondary structure motif in which two consecutive adenine residues occupy the strand opposite a single uridine, creating the potential to form one of two different A.U base pairs while leaving the other adenine unpaired or bulged. During the splicing of introns out of pre-mRNA, the 2'-OH of the bulged adenine participates in the transesterification reaction at the 5'-exon and forms the branch-point residue of the lariat intermediate. Either adenine may act as the branch-point residue in mammals, but the 3'-proximal adenine does so preferentially. When bound to phage GA coat protein, the bulged adenine loops out of the helix and occupies a binding pocket on the surface of the protein, forming a nucleation complex for phage assembly. The coat protein can bind helices with bulged adenines at either position, but the 3'-proximal site binds with greater affinity. We have studied this RNA motif in a 21 nucleotide hairpin containing a GA coat protein-binding site whose four nucleotide loop has been replaced by a more stable loop from the related phage Ms2. Using heteronuclear NMR spectroscopy, we have determined the structure of this hairpin to an overall precision of 2.0 A. Both adenine bases stack into the helix, and while all available NOE and coupling constant data are consistent with both possible A.U base pairs, the base pair involving the 5'-proximal adenine appears to be the major conformation. The 3'-proximal bulged adenine protonates at unusually high pH, and to account for this, we propose a model in which the protonated adenine is stabilized by a hydrogen bond to the uridine O2 of the A.U base pair. The 2'-OH of the bulged adenine adopts a regular A-form helical geometry, suggesting that in order to participate in the splicing reaction, the conformation of the branch-point helix in the active spliceosome may change from the conformation described here. Thus, while the adenine site preferences of the spliceosome and of phage GA may be due to protein factors, the preferred adenine is predisposed in the free RNA to conformational rearrangement involved in formation of the active complexes.     

Solution structure of the tobramycin-RNA aptamer complex

We have solved the solution structure of the aminoglycoside antibiotic tobramycin complexed with a stem-loop RNA aptamer. The 14 base loop of the RNA aptamer 'zippers up' alongside the attached stem through alignment of four mismatches and one Watson-Crick pair on complex formation. The tobramycin inserts into the deep groove centered about the mismatch pairs and is partially encapsulated between its floor and a looped out guanine base that flaps over the bound antibiotic. Several potential intermolecular hydrogen bonds between the charged NH3 groups of tobramycin and acceptor atoms on base pair edges and backbone phosphates anchor the aminoglycoside antibiotic within its sequence/structure specific RNA binding pocket.     

Structural features of the DNA hairpin d(ATCCTA-GTTA-TAGGAT): formation of a G-A base pair in the loop

The three-dimensional structure of the hairpin formed by d(ATCCTA-GTTA-TAGGAT) has been determined by means of two-dimensional NMR studies, distance geometry and molecular dynamics calculations. The first and the last residues of the tetraloop of this hairpin form a sheared G-A base pair on top of the six Watson-Crick base pairs in the stem. The glycosidic torsion angles of the guanine and adenine residues in the G-A base pair reside in the anti and high- anti domain ( approximately -60 degrees ) respectively. Several dihedral angles in the loop adopt non-standard values to accommodate this base pair. The first and second residue in the loop are stacked in a more or less normal helical fashion; the fourth loop residue also stacks upon the stem, while the third residue is directed away from the loop region. The loop structure can be classified as a so-called type-I loop, in which the bases at the 5'-end of the loop stack in a continuous fashion. In this situation, loop stability is unlikely to depend heavily on the nature of the unpaired bases in the loop. Moreover, the present study indicates that the influence of the polarity of a closing A.T pair is much less significant than that of a closing C.G base pair.     

Improved parameters for the prediction of RNA hairpin stability

Thermodynamic parameters are reported for hairpin formation in 1 M NaCl by RNA sequences of the type GGXANmAYCC, where XY is the set of four Watson-Crick base pairs and the underlined loop sequences are three to nine nucleotides. A nearest neighbor analysis of the data indicates the free energy of loop formation at 37 degrees C is dependent upon loop size and closing base pair. The model previously developed to predict the stability for RNA hairpin loops (n > 3) includes contributions from the size of the loop, the identity of the closing base pair, the free energy increment (deltaGo(37mm)) for the interaction of the closing base pair with the first mismatch and an additional stabilization term for GA and UU first mismatches [Serra, M. J., Axenson, T. J., & Turner, D. H. (1994) Biochemistry 33, 14289]. The results presented here allow improvements in the parameters used to predict RNA hairpin stability. For hairpin loops of n = 4-9, deltaGo(37iL)(n) is 4.9, 5.0, 5.0, 5.0, 4.9, and 5.5 kcal/mol, respectively, and the penalty for hairpin closure by AU or UA is +0.6 kcal/mol. deltaGo(37iL)(n) is the free energy for initiating a loop of n nucleotides. The model for predicting hairpin loop stability for loops larger than three becomes deltaGo(37L)(n) = deltaGo(37iL)(n) + deltaGo(37mm) + 0.6(if closed by AU or UA) - 0.7(if first mismatch is GA or UU). Hairpin loops of three are modeled as independent of loop sequence with deltaGo(37iL)(3) = 4.8 and the penalty for AU closure of +0.6 kcal/mol. Thermodynamic parameters for hairpin formation in 1 M NaCl for 11 naturally occurring RNA hairpin sequences are reported. The model provides good agreement with the measured values for both T(M) (within 10 degrees C of the measured value) and deltaGo(37) (within 0.8 kcal/mol of the measured value) for hairpin formation. In general, the nearest neighbor model allows prediction of RNA hairpin stability to within 5-10% of the experimentally measured values.