Solution structure of an RNA internal loop with three consecutive sheared GA pairs

Internal loops in RNA are important for folding and function. Many folding motifs are internal loops containing GA base pairs, which are usually thermodynamically stabilizing, i.e., contribute favorable free energy to folding. Understanding the sequence dependence of folding stability and structure in terms of molecular interactions, such as hydrogen bonding and base stacking, will provide a foundation for predicting stability and structure. Here, we report the NMR structure of the oligonucleotide duplex, 5'GGUGGAGGCU3'/3'PCCGAAGCCG5' (P = purine), containing an unusually stable and relatively abundant internal loop, 5'GGA3'/3'AAG5'. This loop contains three consecutive sheared GA pairs (trans Hoogsteen/Sugar edge AG) with separate stacks of three G's and three A's in a row. The thermodynamic consequences of various nucleotide substitutions are also reported. Significant destabilization of approximately 2 kcal/mol at 37 degrees C is found for substitution of the middle GA with AA to form 5'GAA3'/3'AAG5'. This destabilization correlates with a unique base stacking and hydrogen-bonding network within the 5'GGA3'/3'AAG5' loop. Interestingly, the motifs, 5'UG3'/3'GA5' and 5'UG3'/3'AA5', have stability similar to 5'CG3'/3'GA5' even though UG and UA pairs are usually less stable than CG pairs. Consecutive sheared GA pairs in the 5'GGA3'/3'AAG5' loop are preorganized for potential tertiary interactions and ligand binding.     

Consecutive GA pairs stabilize medium-size RNA internal loops

Internal loops in RNA are important for folding and function. Consecutive noncanonical pairs can form in internal loops having at least two nucleotides on each side. Thermodynamic and structural insights into such internal loops should improve approximations for their stabilities and predictions of secondary and three-dimensional structures. Most natural internal loops are purine rich. A series of oligoribonucleotides that form purine-rich internal loops of 5-10 nucleotides, including kink-turn loops, were studied by UV melting, exchangeable proton and phosphorus NMR. Three consecutive GA pairs with the motif 5' Y GGA/3' R AAG or GGA R 3'/AAG Y 5' (i.e., 5' GGA 3'/3' AAG 5' closed on at least one side with a CG, UA, or UG pair with Y representing C or U and R representing A or G) stabilize internal loops having 6-10 nucleotides. Certain motifs with two consecutive GA pairs are also stabilizing. In internal loops with three or more nucleotides on each side, the motif 5' U G/3' G A has stability similar to 5' C G/3' G A. A revised model for predicting stabilities of internal loops with 6-10 nucleotides is derived by multiple linear regression. Loops with 2 x 3 nucleotides are predicted well by a previous thermodynamic model.     

The NMR structure of an internal loop from 23S ribosomal RNA differs from its structure in crystals of 50s ribosomal subunits

Internal loops play an important role in structure and folding of RNA and in recognition of RNA by other molecules such as proteins and ligands. An understanding of internal loops with propensities to form a particular structure will help predict RNA structure, recognition, and function. The structures of internal loops 5' 1009CUAAG1013 3'/3' 1168GAAGC1164 5' and 5' 998CUAAG1002 3'/3' 1157GAAGC1153 5' from helix 40 of the large subunit rRNA in Deinococcus radiodurans and Escherichia coli, respectively, are phylogenetically conserved, suggesting functional relevance. The energetics and NMR solution structure of the loop were determined in the duplex 5' 1GGCUAAGAC9 3'/3' 18CCGAAGCUG10 5'. The internal loop forms a different structure in solution and in the crystal structures of the ribosomal subunits. In particular, the crystal structures have a bulged out adenine at the equivalent of position A15 and a reverse Hoogsteen UA pair (trans Watson-Crick/Hoogsteen UA) at the equivalent of U4 and A14, whereas the solution structure has a single hydrogen bond UA pair (cis Watson-Crick/sugar edge A15U4) between U4 and A15 and a sheared AA pair (trans Hoogsteen/sugar edge A14A5) between A5 and A14. There is cross-strand stacking between A6 and A14 (A6/A14/A15 stacking pattern) in the NMR structure. All three structures have a sheared GA pair (trans Hoogsteen/sugar edge A6G13) at the equivalent of A6 and G13. The internal loop has contacts with ribosomal protein L20 and other parts of the RNA in the crystal structures. These contacts presumably provide the free energy to rearrange the base pairing in the loop. Evidently, molecular recognition of this internal loop involves induced fit binding, which could confer several advantages. The predicted thermodynamic stability of the loop agrees with the experimental value, even though the thermodynamic model assumes a Watson-Crick UA pair.     

NMR structure of a 4 x 4 nucleotide RNA internal loop from an R2 retrotransposon: identification of a three purine-purine sheared pair motif and comparison to MC-SYM predictions

The NMR solution structure is reported of a duplex, 5'GUGAAGCCCGU/3'UCACAGGAGGC, containing a 4 × 4 nucleotide internal loop from an R2 retrotransposon RNA. The loop contains three sheared purine-purine pairs and reveals a structural element found in other RNAs, which we refer to as the 3RRs motif. Optical melting measurements of the thermodynamics of the duplex indicate that the internal loop is 1.6 kcal/mol more stable at 37°C than predicted. The results identify the 3RRs motif as a common structural element that can facilitate prediction of 3D structure. Known examples include internal loops having the pairings: 5'GAA/3'AGG, 5'GAG/3'AGG, 5'GAA/3'AAG, and 5'AAG/3'AGG. The structural information is compared with predictions made with the MC-Sym program.     

Stable sheared A.C pair in DNA hairpins

Single-residue d(Pu1NPu2) (Pu1.Pu2=G.A, G.G or A.A) hairpin loops can be stably closed by sheared purine.purine pairs. These special motifs have been found in several important biological systems. We now extend these loop-closing base-pairs to a sheared purine. pyrimidine (A.C) pair at a neutral pH condition. High-resolution NMR spectroscopy, distance geometry, and molecular dynamics methods were used to study d(GTACANCGTAC) oligomers. Numerous idiosyncratic nuclear Overhauser enhancements, especially those across the A.C base-pair between C4NH2left and right arrow AH1', C4NH2left and right arrow AH2, and CH5left and right arrow AH2 proton pairs, clearly define the novel sheared nature of the closing A.C base-pair. This novel base-pair is possibly present in several biological systems and in two single-stranded DNA aptamers selected from oligonucleotide libraries.     

Molecular recognition in purine-rich internal loops: thermodynamic,structural, and dynamic consequences of purine for adenine substitutions in 5'(rGGCAAGCCU)2

The contribution of amino groups to the thermodynamics, structure, and dynamics of tandem A.A mismatches is investigated by substitution of purine (P) for adenine (A) within the RNA duplex, 5'(rGGCAAGCCU)(2), to give 5'(rGGCPAGCCU)(2), 5'(rGGCAPGCCU)(2), and 5'(rGGCPPGCCU)(2). The 5'(rGGCAAGCCU)(2) duplex has sheared A(anti).A(anti) (A.A trans Hoogsteen/Sugar-edge) pairs in which the A5 amino group is involved in hydrogen bonds but the A4 amino group is not [Znosko, B. M., Burkard, M. E., Schroeder, S. J., Krugh, T. R., and Turner, D. H. (2002) Biochemistry 41, 14969-14977]. In comparison to 5'(rGGCAAGCCU)(2), replacing the amino group of A4 with a hydrogen stabilizes the duplex by 1.3 kcal/mol, replacement of the A5 amino group destabilizes the duplex by 0.6 kcal/mol, and replacement of both A4 and A5 amino groups destabilizes the duplex by 0.8 kcal/mol. In NMR structures, the P.A noncanonical pairs of the 5'(rGGCPAGCCU)(2) duplex have a sheared anti-anti structure (P.A trans Hoogsteen/Sugar-edge) with P4.A5 interstrand hydrogen bonding and A5 bases that interstrand stack, similar to the structure of 5'(rGGCAAGCCU)(2). In contrast, the A.P pairs of the 5'(rGGCAPGCCU)(2) duplex have a face-to-face conformation (A.P trans Watson-Crick/Watson-Crick) with intrastrand stacking resembling typical A-form geometry. Although the P5 bases in 5'(rGGCPPGCCU)(2) are involved in an interstrand stack, the loop region is largely undefined. The results illustrate that both hydrogen-bonded and non-hydrogen-bonded amino groups play important roles in determining the thermodynamic, structural, and dynamic characteristics of purine rich internal loops.     

Structural features and thermodynamics of the J4/5 loop from the Candida albicans and Candida dubliniensis group I introns

The J4/5 loop of group I introns has tertiary interactions with the P1 helix that position the P1 substrate for the self-splicing reaction. The J4/5 loop of Candida albicans and Candida dubliniensis, 5'GAAGG3'/3'UAAUU5', potentially contains two A.A pairs flanked by one G.U pair on one side and two G.U pairs on the other side. Results from optical melting, nuclear magnetic resonance spectroscopy, and functional group substitution experiments with a mimic of the C. albicans and C. dubliniensis J4/5 loop are consistent with the adenosines forming tandem sheared A.A pairs with a cross-strand stack and only the G.U pair not adjacent to an A.A pair forming a static wobble G.U pair. The two G.U pairs adjacent to the tandem A.A pairs are likely in a dynamic equilibrium between multiple conformations. Although Co(NH(3))(6)(3+) stabilizes the loop by several kilocalories per mole at 37 degrees C, addition of Mg(2+) or Co(NH(3))(6)(3+) has no effect on the structure of the loop. The tandem G.U pairs provide a pocket of negative charge for Co(NH(3))(6)(3+) to bind. The results contribute to understanding the structure and dynamics of purine-rich internal loops and potential G.U pairs adjacent to internal loops.     

RNA hairpin loop stability depends on closing base pair.

Thermodynamic parameters are reported for hairpin formation in 1 M NaCl by RNA sequences of the type GGXAUAAUAYCC, where X and Y are CG, GC, AU, UA, GU, or UG. A nearest neighbor analysis of the data indicates the free energy change for loop formation at 37 degrees C, delta degrees Gl,37, averages 3.4 kcal/mol for hairpin loops closed with C.G, G.C, and G.U pairs. In contrast, delta G degree l,37 averages 4.6 kcal/mol for loops closed with A.U, U.A, or U.G pairs. Thus the stability of an RNA hairpin depends on the closing base pair. The hairpin with a GA mismatch that is formed by GGCGUAAUAGCC is more stable than the corresponding hairpin with an AA mismatch. Thus hairpin stability also depends on loop sequence. These effects are not included in current algorithms for prediction of RNA structure from sequence.     

Sheared Aanti.Aanti base pairs in a destabilizing 2 x 2 internal loop: the NMR structure of 5'(rGGCAAGCCU)2

The 5'(rGGCAAGCCU)(2) duplex contains tandem A.A pairs. The three-dimensional structure of the 5'(rGGCAAGCCU)(2) duplex was modeled by molecular dynamics and energy minimization with NMR-derived distance and dihedral angle restraints. Although the 5'(rCAAG)(2) loop is thermodynamically destabilizing by 1.1 kcal/mol, the tandem A.A pairs adopt a predominant conformation: a sheared anti-anti (A.A trans Hoogsteen/Sugar-edge) alignment similar to that observed in the crystal structure of the P4-P6 domain of the Tetrahymena thermophila intron [Cate, J. H., Gooding, A. R., Podell, E., Zhou, K., Golden, B. L., Kundrot, C. E., Cech, T. R., and Doudna, J. A. (1996) Science 273, 1678-1685]. The NMR-derived structure of the 5'(rGGCAAGCCU)(2) duplex exhibits cross-strand hydrogen bonds from N3 of A4 to an amino hydrogen of A5 and from the 2' oxygen of the A4 sugar to the other amino hydrogen of A5. An intrastrand hydrogen bond is formed from the 2' OH hydrogen of A4 to O5' of A5. The cross-strand A5 bases are stacked. The Watson-Crick G-C regions are essentially A-form. The sheared anti-anti (A.A trans Hoogsteen/Sugar-edge) alignment provides potential contact sites for tertiary interactions and, therefore, is a possible target site for therapeutics. Thus, thermodynamically destabilizing internal loops can be preorganized for tertiary interactions or ligand binding.     

Cleavage of single stranded oligonucleotides by EcoRI restriction endonuclease.

The 31mer 5'-TCA ACG CTA GAA TTC GGA TCC ATC GCT TGG T, the complementary 33mer 5'-CCA AGC GAT GGA TCC GAA TTC TAG CGT TGA GAT, the 40mer 5'-GGC CAG GAT GGT GAA GAA TTC GAT CCG GTA CGT AGC TAA G, and the complementary 42mer 5'-TAC TTA GCT ACG TAC CGG ATC GAA TTC TTC ACC ATC CTG GCC were synthesized and their reactivity towards EcoRI was studied. It was found that the 31mer and the 40mer were cleaved at a comparable rate to the 31mer-33mer hybrid and the 40mer-42mer hybrid, respectively. The rate of cleavage of the 33mer and the 42mer was an order of magnitude lower. To rule out possible intermolecular duplex formation, the 33mer was immobilized on cellulose by ligation and labeled with alpha 32P-dCTP using Klenow fragment of E. coli DNA polymerase. EcoRI cleaved this immobilized oligomer into specific fragments.     

Analysis of the functional role of a G.A sheared base pair by in vitro genetics

A classical genetic strategy has been combined with an in vitro selection method to search for functional interactions between the two domains of the hairpin ribozyme. G(21) is located within internal loop B; it is proposed to form a sheared base pair with A(43) across loop B and to bind a Mg(2+) ion. Both nucleotides are important for ribozyme function, and G.A sheared base pairs are a very widespread motif in structured RNA. We took advantage of its presence in the hairpin ribozyme to study its functional role. Pseudorevertants, in which the loss of G(21) was compensated by mutations at other positions, were isolated by in vitro selection. The vast majority of G(21) revertants contained substitutions within domain A, pointing to functional communication between specific sites within the two domains of the hairpin ribozyme. The possibility of a direct or redundant contacts is supported by electrophoretic mobility shift studies showing that a complex formed between domain B of the ribozyme and the substrate was disrupted and restored by base substitutions that have analogous effects on catalytic activity. The functional significance of this complex, the role of the nucleotides involved, and the basis for magnesium ion requirement is discussed.     

Molecular Detection of Colletotrichum lindemuthianum by Duplex PCR

A duplex PCR technique was developed to detect the pathogenic fungus Colletotrichum lindemuthianum infection in the tissues of common bean. Based on the differences of 24 internal transcribed spacer, DNA sequences of Colletotrichum spp. retrieved from GeneBank database, one pair of specific primers of CY1/CY2 (CY1: 5'-CTT TGT GAA CAT ACC TAA CC-3'; CY2: 5'-GGT TTT ACG GCA GGA GTG-3'), was designed. The CY1/CY2 primers amplified a single PCR product of 442 bp only from C. lindemuthianum and Colletotrichum orbiculare , not from any other tested species. By using random amplification of polymorphic DNA technique, a product closely associated with C. lindemuthianum was generated. This product was cloned, sequenced and used for designing a species-specific primers of CD1/CD2 (CD1: 5'-ACC TGG ACA CAT AAG TCA AAG-3'; CD2: 5'-CAA CAA TGC CAG TAT CAG AG-3'). The CD1/CD2 primers could distinguish C. lindemuthianum from C. orbiculare by a 638 bp PCR band. A duplex PCR method, combining both primers of CY1/CY2 and CD1/CD2, was used to detect C. lindemuthianum infection. The sensitivity of the detection with this PCR method was 1 pg of pure genomic DNA from the pathogen. Therefore, the PCR-based methods could be used for accurate and rapid detection of C. lindemuthianum from common bean.     

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.     

Formation of sheared G:A base pairs in an RNA duplex modelled after ribozymes, as revealed by NMR.

The thermal stability and structure of an RNA duplex, r(GGACGAGUCC)2, the base sequence of which was modelled after both a hammerhead ribozyme and a lead ribozyme, were studied by CD and NMR. We previously demonstrated that the corresponding DNA duplex, d(GGACGAGTCC)2, formed unique 'sheared' G:A base pairs, where an amino proton, instead of an imino proton, of G is involved in the hydrogen bonding, and G and A bases are arranged 'side by side' instead of 'head to head' (Nucleic Acids Res. (1993) 21, 5418-5424). CD melting profiles showed that the RNA duplex is thermally more stable than the corresponding DNA duplex. NMR studies revealed that sheared G:A base pairs are formed in the RNA duplex, too, although the overall structure of the RNA is the A form, which differs from the B form taken on by the corresponding DNA. A model building study confirmed that sheared G:A base pairs can be accommodated in the double helical structure of the A form. A difference between the RNA and DNA duplexes in the stacking interaction involving G:A mismatch bases is also suggested. The demonstration that sheared G:A base pairs can be formed not only in DNA but also in RNA suggests that this base pairing plays an important role regarding the RNA structure.     

Thermodynamic stability and structural features of the J4/5 loop in a Pneumocystis carinii group I intron

The J4/5 loop of the group I intron in the mouse-derived fungal pathogen Pneumocystis carinii is the docking site for the first step of the RNA-catalyzed self-splicing reaction and thus is a model of a potential drug target. This purine-rich asymmetric internal loop, 5'GGAAG/3'UAGU, is also thermodynamically more stable than other internal loops with two GU closing pairs and three nucleotides opposite two nucleotides. The results from optical melting, nuclear magnetic resonance spectroscopy, and functional group substitution experiments suggest that the GU closing pairs form and that sheared GA pairs form in the internal loop. The NMR spectra show evidence of conformational dynamics, and several GA pairings are possible. Thus, this dynamic loop presents several possible structures for potential binding of drugs that target group I self-splicing introns. The results also contribute to understanding the structural and dynamic basis for the function and thermodynamic stability of this loop.     

Nuclear magnetic resonance spectroscopy and molecular modeling reveal that different hydrogen bonding patterns are possible for G.U pairs: one hydrogen bond for each G.U pair in r(GGCGUGCC)(2) and two for each G.U pair in r(GAGUGCUC)(2)

G.U pairs occur frequently and have many important biological functions. The stability of symmetric tandem G.U motifs depends both on the adjacent Watson-Crick base pairs, e.g., 5'G > 5'C, and the sequence of the G.U pairs, i.e., 5'-UG-3' > 5'-GU-3', where an underline represents a nucleotide in a G.U pair [Wu, M., McDowell, J. A., and Turner, D. H. (1995) Biochemistry 34, 3204-3211]. In particular, at 37 degrees C, the motif 5'-CGUG-3' is less stable by approximately 3 kcal/mol compared with other symmetric tandem G.U motifs with G-C as adjacent pairs: 5'-GGUC-3', 5'-GUGC-3', and 5'-CUGG-3'. The solution structures of r(GAGUGCUC)(2) and r(GGCGUGCC)(2) duplexes have been determined by NMR and restrained simulated annealing. The global geometry of both duplexes is close to A-form, with some distortions localized in the tandem G.U pair region. The striking discovery is that in r(GGCGUGCC)(2) each G.U pair apparently has only one hydrogen bond instead of the two expected for a canonical wobble pair. In the one-hydrogen-bond model, the distance between GO6 and UH3 is too far to form a hydrogen bond. In addition, the temperature dependence of the imino proton resonances is also consistent with the different number of hydrogen bonds in the G.U pair. To test the NMR models, U or G in various G.U pairs were individually replaced by N3-methyluridine or isoguanosine, respectively, thus eliminating the possibility of hydrogen bonding between GO6 and UH3. The results of thermal melting studies on duplexes with these substitutions support the NMR models.     

Nearest neighbor parameters for inosine x uridine pairs in RNA duplexes

An enzyme family known as adenosine deaminases that act on RNA (ADARs) catalyzes adenosine deamination in RNA. ADARs act on RNA that is largely double-stranded and convert adenosine to inosine, resulting, in many cases, in an I x U pair. Thermodynamic parameters derived from optical melting studies are reported for a series of 14 oligoribonucleotides containing single I x U pairs adjacent to Watson-Crick pairs. In order to determine unique linearly independent nearest neighbor parameters for I x U pairs, four duplexes containing 3'-terminal I x U pairs and four duplexes containing 5'-terminal I x U pairs have also been thermodynamically characterized. This data was combined with previously published data of seven duplexes containing internal, terminal, or tandem I x U pairs from Strobel et al. [Strobel, S. A., Cech, T. R., Usman, N., and Beigelman, L. (1994) Biochemistry 33, 13824-13838] and Serra et al. [Serra, M. J., Smolter, P. E., and Westhof, E. (2004) Nucleic Acids Res. 32, 1824-1828]. On average, a duplex with an internal I x U pair is 2.3 kcal/mol less stable than the same duplex with an A-U pair, however, a duplex with a terminal I x U pair is 0.8 kcal/mol more stable than the same duplex with an A-U pair. Although isosteric with a G-U pair, on average, a duplex with an internal I x U pair is 1.9 kcal/mol less stable than the same duplex with a G-U pair, however, a duplex with a terminal I x U pair is 0.9 kcal/mol more stable than the same duplex with a G-U pair. Duplexes with tandem I x U pairs are on average 5.9 and 3.8 kcal/mol less stable than the same duplex with tandem A-U or tandem G-U pairs, respectively. Using the combined thermodynamic data and a complete linear least-squares fitting routine, nearest neighbor parameters for all nearest neighbor combinations of I x U pairs and an additional parameter for terminal I x U pairs have been derived.     

Molecular recognition by the Candida albicans group I intron: tertiary interactions with an imino G.A pair facilitate binding of the 5' exon and lower the KM for guanosine

A G.A pair at position -5 in the P1 helix of the Candida albicans ribozyme contributes to tertiary binding of the 5' exon substrate [Disney, M. D., Haidaris, C. G., and Turner, D. H. (2001) Biochemistry 40, 6507-6519]. Here, the G in the G.A pair is replaced with inosine (I) in both semisynthetic ribozymes and oligonucleotide mimics of the internal guide sequence. Comparisons of oligonucleotide binding affinity for these and other sequences indicate that the G.A pair is in an imino conformation where the exocyclic amine of G contributes approximately 1.4 kcal/mol to tertiary interactions that help dock the ribozyme's P1 helix. Furthermore, replacement of the G.A pair with a G-C pair produces less favorable interactions with the 2'-hydroxyl group at the -3 position and a less favorable K(M) for pG in a ribozyme-catalyzed transesterification reaction. These results are also consistent with the G.A pair promoting docking of the P1 helix into the catalytic core. Evidently, tertiary interactions with the exocyclic amino group of a G in a single G.A pair can increase the equilibrium constant for tertiary folding of RNA by roughly 10-fold at 37 degrees C. Results with a G.U or G.G pair replacing the G.A pair at the -5 position suggest similar tertiary interactions with these pairs.