NMR reveals the absence of hydrogen bonding in adjacent UU and AG mismatches in an isolated internal loop from ribosomal RNA.

NMR studies provide insights into structural features of internal loops. These insights can be combined with thermodynamic studies to generate models for predicting structure and energetics. The tandem mismatch internal loop, 5'GUGG3'(3'CUAC5'), has been studied by NMR. The NMR structure reveals an internal loop with no hydrogen bonding between the loop bases and with the G in the AG mismatch flipped out of the helix. The sequence of this internal loop is highly conserved in rRNA. The loop is located in the large ribosomal subunit and is part of a conserved 58-nt fragment that is the binding domain of ribosomal protein L11. Structural comparisons between variants of this internal loop in crystal structures of the 58-nt domain complexed with L11 protein and of the large ribosomal subunit (LSU) suggest that this thermodynamically destabilizing internal loop is partially preorganized for tertiary interactions and for binding L11. A model for predicting the base pairing and free energy of 2 x 2 nucleotide internal loops with a purine-purine mismatch next to a pyrimidine-pyrimidine mismatch is proposed on the basis of the present NMR structure and previously reported thermodynamics.

Stacking effects on local structure in RNA: changes in the structure of tandem GA pairs when flanking GC pairs are replaced by isoG-isoC pairs.

The Watson-Crick-like isoG-isoC (iGiC) pair, with the amino and carbonyl groups transposed relative to the Watson-Crick GC pair, provides an expanded alphabet for understanding interactions that shape nucleic acid structure. Here, thermodynamic stabilities of tandem GA pairs flanked by iGiC pairs are reported along with the NMR structures of the RNA self-complementary duplexes (GCiGGAiCGCA)2 and (GGiCGAiGCCA)2. A sheared GA pairing forms in (GCiGGAiCGCA)2, and an imino GA pairing forms in (GGiCGAiGCCA)2. The structures contrast with the formation of tandem imino and sheared GA pairs flanked by GC pairs in the RNA self-complementary duplexes (GCGGACGC)2 and (GGCGAGCC)2, respectively. In both iGiC duplexes, Watson-Crick-like hydrogen bonds are formed between iG and iC, and iGiC substitutions result in less favorable loop stability. The results provide benchmarks for testing computations of molecular interactions that shape RNA three-dimensional structure.

NMR structures of (rGCUGAGGCU)2 and (rGCGGAUGCU)2: probing the structural features that shape the thermodynamic stability of GA pairs.

The NMR structures of [see text] and [see text] are reported. The internal loop, [see text], is about 2 kcal/mol more stable than [see text] at 37 degrees C. The duplexes assemble into similar global folds characterized by the formation of tandem sheared GA pairs. The different stabilities of the loops are accompanied by differences in the local structure of the closing GU pairs. In the [see text] internal loop, the GU pairs form canonical wobble configurations with two hydrogen bonds, whereas in [see text], the GU pairs form a single hydrogen bond involving the amino group, GH22, and the carbonyl group, UO4. This pairing is similar to the GU closing pair of the 690 hairpin loop found in E. coli 16S rRNA. The [see text] and [see text] structures reveal how the subtle interplay between stacking and hydrogen bonding determines sequence dependent conformation and thermodynamic stability. Thus, this work provides structural and thermodynamic benchmarks for theoreticians in the ongoing effort to understand the sequence dependence of RNA physicochemical properties.

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.

An alternating sheared AA pair and elements of stability for a single sheared purine-purine pair flanked by sheared GA pairs in RNA.

A previous NMR structure of the duplex 5'GGU GGA GGCU/PCCG AAG CCG5' revealed an unusually stable RNA internal loop with three consecutive sheared GA pairs. Here, we report NMR studies of two duplexes, 5'GGU GGA GGCU/PCCA AAG CCG5' (replacing the UG pair with a UA closing pair) and 5'GGU GAA GGCU/PCCG AAG CCG5' (replacing the middle GA pair with an AA pair). An unusually stable loop with three consecutive sheared GA pairs forms in the duplex 5'GGU GGA GGCU/PCCA AAG CCG5'. The structure contrasts with that reported for this loop in the crystal structure of the large ribosomal subunit of Deinococcus radiodurans [Harms, J., Schluenzen, F., Zarivach, R., Bashan, A., Gat, S., Agmon, I., Bartels, H., Franceschi, F., and Yonath, A. (2001) Cell 107, 679-688]. The middle AA pair in the duplex 5'GGU GAA GGCU/PCCG AAG CCG5' rapidly exchanges orientations, resulting in alternative base stacking and pseudosymmetry with exclusively sheared pairs. The U GAA G/G AAG C internal loop is 2.1 kcal/mol less stable than the U GGA G/G AAG C internal loop at 37 degrees C. Structural, energetic, and dynamic consequences upon functional group substitutions within related 3 x 3 and 3 x 6 internal loops are also reported.

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.

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.

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.

NMR studies of DNA single strands and DNA:RNA hybrids with and without 1-propynylation at C5 of oligopyrimidines.

The 1-propynylation at C5 of consecutive pyrimidines in DNA can enhance DNA:RNA hybrid stability at 37 degrees C by over 1 kcal/mol of substitution [Barnes, T. W., III; Turner, D. H. J. Am. Chem. Soc.2001, 123, 4107-4118]. To provide information on the structural consequences of propynylation, two-dimensional NMR spectroscopy was used to study the structures of several oligonucleotides. Intraresidue nuclear Overhauser effect spectroscopy cross peaks were observed at 30 degrees C and a 200 ms mixing time in the H6-H1' region for 5'(dC(P)C(P)U(P)C(P)C(P)U(P)U(P)) (ssPrODN) but not for 5'(dCCUCCUU) (ssODN), suggesting preorganization of the propynylated single strand. NMR structures of the duplexes 5'(dC(P)C(P)U(P)C(P)C(P)U(P)U(P))3':3'(rGAGGAGGAAAU)5' (PrODN:RNA), 5'(dCC(P)U(P)C(P)C(P)U(P)U(P))3':3'(rGAGGAGGAAAU)5' (sPrODN1:RNA), and 5'(dCCUCCUU)3':3'(rGAGGAGGAAAU)5' (ODN:RNA) indicate that their global structures are almost identical. The NMR data, however, suggest that the 5'-end of sPrODN1:RNA is more dynamic than that of PrODN:RNA. In the propynylated duplexes, the propyne group stacks on the aromatic ring of the 5'-base and extends into the major groove. The results suggest that the increased stability of the propynylated duplexes is caused by preorganization of the propynylated single strand and different interactions in the double strand. The propynyl group provides volume exclusion, enhanced stacking, and possibly different solvation.

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.

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.

Solution Structure of a Nonpolar, Non-Hydrogen-Bonded Base Pair Surrogate in DNA.

We describe the structure in aqueous solution of a DNA duplex containing a base pair that is structurally analogous to A-T but which lacks hydrogen bonds. Base analogues F (a nonpolar isostere of thymine) and Z (a nonpolar isostere of adenine) are paired opposite one another in a 12 base pair duplex. The sequence context is the binding site of recently studied transcription factor hSRY. The Z-F pair has been shown to be replicated surprisingly well and selectively by DNA polymerase enzymes, considering that it is destabilizing and lacks Watson-Crick hydrogen bonds. The enzymatic studies led to the suggestion that part of the functional activity arises because the pair resembles a natural one in geometry. The present results show that, despite the absence of Watson-Crick hydrogen bonds, the Z-F pair structurally resembles an A-T pair in the same context. This lends support to the proposal that shape matching is an important component in replication, and suggests the general utility of using Z-F as a nonpolar replacement for A-T in probing protein-DNA interactions.