Solution Structure of (rGCGGACGC)₂ by Two-Dimensional NMR and the Iterative Relaxation Matrix Approach

Abstract

The three-dimensional solution structure of the RNA self-complementary duplex [sequence: see text] was derived from two-dimensional NMR and the iterative relaxation matrix approach. Each GA mismatch forms two hydrogen bonds: A-NH6 to G-O6 and A-N1 to G-NH1 (imino). This is the first three-dimensional RNA structure with imino hydrogen-bonded tandem GA mismatches. This GA structure is totally different from the sheared tandem GA structure in [sequence: see text] which also has two hydrogen bonds: A-N7 to G-NH2 and A-NH6 to G-N3 [SantaLucia, J., Jr., & Turner, D. H. (1993) Biochemistry 32, 12612-12623]. In particular, the sheared and imino GA mismatches produce a narrowing and widening of the backbone, respectively. The results show that substitutions of Watson-Crick base pairs can have dramatic effects on the three-dimensional structures of adjacent non-Watson-Crick paired regions; i.e., the structure depends on sequence context. Thus compensating substitutions in site-directed mutagenesis experiments may not always restore biological activities.

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

“…1,4,11 Tandem imino GA (cis Watson-Crick/Watson-Crick A-G) pairs (Figure 1) form in the symmetric context (GGAC) 2 with loop free energy of −2.5 kcal/ mol. 1,4,9 These results suggest that base stacking interactions (e.g., Coulombic and overlap) determine the hydrogen bonding patterns of RNA tandem GA pairs in these internal loops. 7,9,16 Different shapes of GA pairs in turn provide different molecular features, e.g., van der Waals and electrostatic surface, and hydrogen bonding donors and acceptors, for higher order RNA folding and molecular recognition.…”

“…1,4,9 These results suggest that base stacking interactions (e.g., Coulombic and overlap) determine the hydrogen bonding patterns of RNA tandem GA pairs in these internal loops. 7,9,16 Different shapes of GA pairs in turn provide different molecular features, e.g., van der Waals and electrostatic surface, and hydrogen bonding donors and acceptors, for higher order RNA folding and molecular recognition. [13][14][15][17][18][19] The Watson-Crick like iGiC pair, with the amino and carbonyl groups transposed relative to the Watson-Crick GC pair, provides an expanded alphabet [20][21][22][23][24][25][26] for further understanding interactions that shape nucleic acid structure (Figure 1).…”

“…This contrasts with a weak cross-strand A5H2-A5*H1′ cross-peak, with NMR distance ~ 4.1 Å, that was observed in (GCGGACGC) 2 , which contains tandem imino GA pairs with substantial intrastrand base stacking. 9 Several exchangeable proton cross-peaks with potential spin diffusion effects were not used to generate distance restraints.…”

“…The stability [1][2][3][4][5][6][7] and three-dimensional structure [7][8][9][10][11][12][13][14][15] of RNA tandem GA pairs are dependent on sequence context. Tandem sheared GA (trans Hoogsteen/ Sugar edge A-G) pairs ( Figure 1) form with cross-strand base stacking in the symmetric contexts (CGAG) 2 with loop free energy of −0.7 kcal/mol 1,3,8 and (UGAA) 2 with loop free energy of 0.7 kcal/mol.…”

Effects of 3′-terminal phosphates in RNA produced by ribozyme cleavage

“…ited to the cyclic phosphate form because the T 1 s of these same imino protons are enhanced in the RNA form that has both 39-and 29-phosphates at the 39 terminus (Table 1)+ The chemical shift differences between the three forms of the RNA are not completely understood+ The shift differences (the last U-U pair and the last two G-A pairs) of the ribozyme-produced RNA that has the cyclic phosphate may arise from slight conformation alteration at the helical end brought about by the restrained sugar conformation+ This could be explained by the flattening of the ribose pucker brought about by the cyclic phosphate+ This subtle difference may change the stacking geometry of the last three pairs and result in shifts of the terminal G-A iminos+ The cyclic phosphate does not appear to abolish the last base pair as suggested by the similar T m s in all forms of the RNA, but does appear to produce a structural perturbation in the G-A tandem of the core region+ Another structural alteration takes place as a result of the mixture of 29-and 39-phosphates at this helical end+ Effects of 59-phosphates in RNA and DNA can have differing trends depending on the secondary structure+ DNA triplexes seem to be destabilized by a 59-phosphates (Yoon et al+, 1993), whereas RNA duplexes are stabilized by this phosphate (Freier et al+, 1985)+ DNA duplexes do not have their melting temperatures changed appreciably (Yoon et al+, 1993)+ Phosphates at the 39 end in RNA have only small destabilization effects (Freier et al+, 1985)+ Here, all forms of the 39-terminal phosphate act similarly to the 39-phosphate in RNA duplexes, with little thermodynamic alteration in the global melting temperature+ Therefore, the initial resonance broadening observed in the ribozyme-produced SECIS RNA is the result of imino exchange catalysis by a terminal phosphate, and can happen in the absence of observable increased instability at the helix end+ This can be explained by the nature of the GA tandem base pairs+ Both of the G-A pairs in the core region of this RNA are expected to have a "sheared" geometry (Walter et al+, 1994;Walczak et al+, 1996Walczak et al+, , 1998Wu et al+, 1997)+ In this geometry, the iminos of these two Gs are not involved in hydrogen bonding of the pair, and are protected from exchange with solvent only by the two bases above and below+ These iminos are placed in the major groove of the helix, and may be available for catalysis of exchange without opening of the base pair+…”