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A CA(+) pair adjacent to a sheared GA or AA pair stabilizes size-symmetric RNA internal loops.

Chen G, Kennedy SD, Turner DH - Biochemistry (2009)

Bottom Line: The results are consistent with a significantly larger fraction (from approximately 20% at pH 7 to approximately 90% at pH 5.5) of adenines being protonated at the N1 position to form stabilizing wobble CA+ pairs adjacent to a sheared GA or AA pair.No significant pH-dependent stabilization is found for most of the other nearest neighbor combinations involving CA pairs (e.g., CA/AG and AG/CA), which is consistent with the formation of various nonwobble pairs observed in different local sequence contexts in crystal and NMR structures.A revised free-energy model, including stabilization by wobble CA+ pairs, is derived for predicting stabilities of medium-size RNA internal loops.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Rochester, Rochester, New York 14627, USA.

ABSTRACT
RNA internal loops are often important sites for folding and function. Residues in internal loops can have pKa values shifted close to neutral pH because of the local structural environment. A series of RNA internal loops were studied at different pH by UV absorbance versus temperature melting experiments and imino proton nuclear magnetic resonance (NMR). A stabilizing CA pair forms at pH 7 in the CG/AA and CA/AA nearest neighbors when the CA pair is the first noncanonical pair (loop-terminal pair) in 3 x 3 nucleotide and larger size-symmetric internal loops. These CG/AA and CA/AA nearest neighbors, with CA adjacent to a closing Watson-Crick pair, are further stabilized when the pH is lowered from 7 to 5.5. The results are consistent with a significantly larger fraction (from approximately 20% at pH 7 to approximately 90% at pH 5.5) of adenines being protonated at the N1 position to form stabilizing wobble CA+ pairs adjacent to a sheared GA or AA pair. The noncanonical pair adjacent to the GA pair in CG/AA can either stabilize or destabilize the loop, consistent with the sequence-dependent thermodynamics of GA pairs. No significant pH-dependent stabilization is found for most of the other nearest neighbor combinations involving CA pairs (e.g., CA/AG and AG/CA), which is consistent with the formation of various nonwobble pairs observed in different local sequence contexts in crystal and NMR structures. A revised free-energy model, including stabilization by wobble CA+ pairs, is derived for predicting stabilities of medium-size RNA internal loops.

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Base stacking and base pairing involving CA. Base pairs shown in gray lines are closer to the viewer. The Watson−Crick CG, wobble CA+ (the proton from protonation is not shown), and sheared GA pairs shown in a and b are taken from the  segment of the NMR structure of the substrate loop of VS ribozyme (9). The Watson−Crick CG, A N1−C amino single hydrogen-bond AC pair, and sheared GA shown in c and d are taken from  segment of the NMR structure of loop B of a hairpin ribozyme (37). The stacking figures are generated by the 3DNA program (75).
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fig4: Base stacking and base pairing involving CA. Base pairs shown in gray lines are closer to the viewer. The Watson−Crick CG, wobble CA+ (the proton from protonation is not shown), and sheared GA pairs shown in a and b are taken from the segment of the NMR structure of the substrate loop of VS ribozyme (9). The Watson−Crick CG, A N1−C amino single hydrogen-bond AC pair, and sheared GA shown in c and d are taken from segment of the NMR structure of loop B of a hairpin ribozyme (37). The stacking figures are generated by the 3DNA program (75).

Mentions: The thermodynamic stabilization is consistent with the geometric compatibility of and nearest neighbors if the CA pair is protonated and the purine−purine pair is sheared (panels f and g of Figure 1) (3,8,9,36). Solution NMR reveals a protonated wobble CA+ pair adjacent to a sheared GA pair (, sequence in a hairpin ribozyme and VS ribozyme) (see Figure 4 and ), and the pKa of the A (in bold) is about 6.3, according to the pH profile of the chemical shifts of the C2 carbon in adenine (3,8,9). Consistently, the amino protons of A+ (shown in bold) for the symmetric loop resonate at 10.6 ppm at neutral and lower pH (panels e and f of Figure 2 and Figure 3b). In addition, a wobble CA pair forms adjacent to a sheared AA pair (shown in bold) within the J4/5 loop, , in the crystal structure of a group I intron (see ) (36).


A CA(+) pair adjacent to a sheared GA or AA pair stabilizes size-symmetric RNA internal loops.

Chen G, Kennedy SD, Turner DH - Biochemistry (2009)

Base stacking and base pairing involving CA. Base pairs shown in gray lines are closer to the viewer. The Watson−Crick CG, wobble CA+ (the proton from protonation is not shown), and sheared GA pairs shown in a and b are taken from the  segment of the NMR structure of the substrate loop of VS ribozyme (9). The Watson−Crick CG, A N1−C amino single hydrogen-bond AC pair, and sheared GA shown in c and d are taken from  segment of the NMR structure of loop B of a hairpin ribozyme (37). The stacking figures are generated by the 3DNA program (75).
© Copyright Policy - open-access - ccc-price
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2697601&req=5

fig4: Base stacking and base pairing involving CA. Base pairs shown in gray lines are closer to the viewer. The Watson−Crick CG, wobble CA+ (the proton from protonation is not shown), and sheared GA pairs shown in a and b are taken from the segment of the NMR structure of the substrate loop of VS ribozyme (9). The Watson−Crick CG, A N1−C amino single hydrogen-bond AC pair, and sheared GA shown in c and d are taken from segment of the NMR structure of loop B of a hairpin ribozyme (37). The stacking figures are generated by the 3DNA program (75).
Mentions: The thermodynamic stabilization is consistent with the geometric compatibility of and nearest neighbors if the CA pair is protonated and the purine−purine pair is sheared (panels f and g of Figure 1) (3,8,9,36). Solution NMR reveals a protonated wobble CA+ pair adjacent to a sheared GA pair (, sequence in a hairpin ribozyme and VS ribozyme) (see Figure 4 and ), and the pKa of the A (in bold) is about 6.3, according to the pH profile of the chemical shifts of the C2 carbon in adenine (3,8,9). Consistently, the amino protons of A+ (shown in bold) for the symmetric loop resonate at 10.6 ppm at neutral and lower pH (panels e and f of Figure 2 and Figure 3b). In addition, a wobble CA pair forms adjacent to a sheared AA pair (shown in bold) within the J4/5 loop, , in the crystal structure of a group I intron (see ) (36).

Bottom Line: The results are consistent with a significantly larger fraction (from approximately 20% at pH 7 to approximately 90% at pH 5.5) of adenines being protonated at the N1 position to form stabilizing wobble CA+ pairs adjacent to a sheared GA or AA pair.No significant pH-dependent stabilization is found for most of the other nearest neighbor combinations involving CA pairs (e.g., CA/AG and AG/CA), which is consistent with the formation of various nonwobble pairs observed in different local sequence contexts in crystal and NMR structures.A revised free-energy model, including stabilization by wobble CA+ pairs, is derived for predicting stabilities of medium-size RNA internal loops.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Rochester, Rochester, New York 14627, USA.

ABSTRACT
RNA internal loops are often important sites for folding and function. Residues in internal loops can have pKa values shifted close to neutral pH because of the local structural environment. A series of RNA internal loops were studied at different pH by UV absorbance versus temperature melting experiments and imino proton nuclear magnetic resonance (NMR). A stabilizing CA pair forms at pH 7 in the CG/AA and CA/AA nearest neighbors when the CA pair is the first noncanonical pair (loop-terminal pair) in 3 x 3 nucleotide and larger size-symmetric internal loops. These CG/AA and CA/AA nearest neighbors, with CA adjacent to a closing Watson-Crick pair, are further stabilized when the pH is lowered from 7 to 5.5. The results are consistent with a significantly larger fraction (from approximately 20% at pH 7 to approximately 90% at pH 5.5) of adenines being protonated at the N1 position to form stabilizing wobble CA+ pairs adjacent to a sheared GA or AA pair. The noncanonical pair adjacent to the GA pair in CG/AA can either stabilize or destabilize the loop, consistent with the sequence-dependent thermodynamics of GA pairs. No significant pH-dependent stabilization is found for most of the other nearest neighbor combinations involving CA pairs (e.g., CA/AG and AG/CA), which is consistent with the formation of various nonwobble pairs observed in different local sequence contexts in crystal and NMR structures. A revised free-energy model, including stabilization by wobble CA+ pairs, is derived for predicting stabilities of medium-size RNA internal loops.

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