The Stability and Structure of Tandem GA Mismatches in RNA Depend on Closing Base Pairs

Abstract

UV melting and imino proton NMR studies show that the stabilities and structures of tandem GA mismatches in RNA are dependent upon the closing base pairs around these mismatches. Internal loops of sequence 5'XGAY3'3'YAGXS' and 5'XAGY3'3'YGAX5' in the middle of octanucleotides have a range of stabilities over 5 kcal/mol when XY is a Watson-Crick or GU pair. The order of stabilities for these internal loops is 5'-GGAC-3' > UGAG, CGAG > AGAU > UGAA > GGAU. The motifs GGAC, UGAG, and CGAG are stabilizing, while the other GA motifs are destabilizing. The GAGC motif is more stable than CAGG and CGAG, but less stable than GGAC. Chemical shifts for imino protons suggest that the G imino proton of each GA mismatch in 5'-GGAC-3', 5'-GAGC-3', and 5'-CAGG-3' [SantaLucia, J., Jr., Kierzek, R., & Turner, D. H. (1990) Biochemistry 29, 8813-8819] is involved in a hydrogen bond to the base A, whereas in other 5'-XGAY-3' sequences, it is not involved in a hydrogen bond to the base A.

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 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.…”

“…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. 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.…”

“…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.…”

Incorporating chemical modification constraints into a dynamic programming algorithm for prediction of RNA secondary structure

“…For example, an isoenergetic bulged C in 5ЈUGU͞3ЈACCA can occur in two positions. ⌬G°3 7 (special C bulge), Ϫ0.9 Ϯ 0.3 kcal (1 cal ϭ 4.18 J)͞mol, is an empirical bonus applied to bulged C residues adjacent to at least one C. The ⌬G°3 7 bulge initiation for single nucleotide bulges is 3.81 Ϯ 0.08 kcal͞mol.Internal loop free energy parameters are revised on the basis of recent measurements (58-61) and the previously assembled database (8,45,46,(62)(63)(64)(65)(66)(67)(68)(69)). In the program described here, measured values are used when available for 1 ϫ 1, 1 ϫ 2, and Abbreviation: RT, reverse transcription.…”

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+…”