Determination of thermodynamic parameters for HIV DIS type loop-loop kissing complexes

Abstract

The HIV-1 type dimerization initiation signal (DIS) loop was used as a starting point for the analysis of the stability of Watson-Crick (WC) base pairs in a tertiary structure context. We used ultraviolet melting to determine thermodynamic parameters for loop-loop tertiary interactions and compared them with regular secondary structure RNA helices of the same sequences. In 1 M Na+ the loop-loop interaction of a HIV-1 DIS type pairing is 4 kcal/mol more stable than its sequence in an equivalent regular and isolated RNA helix. This difference is constant and sequence independent, suggesting that the rules governing the stability of WC base pairs in the secondary structure context are also valid for WC base pairs in the tertiary structure context. Moreover, the effect of ion concentration on the stability of loop-loop tertiary interactions differs considerably from that of regular RNA helices. The stabilization by Na+ and Mg2+ is significantly greater if the base pairing occurs within the context of a loop-loop interaction. The dependence of the structural stability on salt concentration was defined via the slope of a T(m)/log [ion] plot. The short base-paired helices are stabilized by 8 degrees C/log [Mg2+] or 11 degrees C/log [Na+], whereas base-paired helices forming tertiary loop-loop interactions are stabilized by 16 degrees C/log [Mg2+] and 26 degrees C/log [Na+]. The different dependence on ionic strength that is observed might reflect the contribution of specific divalent ion binding to the preformation of the hairpin loops poised for the tertiary kissing loop-loop contacts.

Figure 1

(A) Schematic representation of the hairpins derived from the HIV-1 DIS hairpin loop used in this study. The kissing loop complex between two hairpins is shown. The loop is composed of nine bases, whereby the six central bases participate in base pairing and are flanked by two adenines on the 5′ side and one adenine on the 3′ side. St1 and St2 stands for stem 1 or stem 2 including flanking adenines, N equals any of the four bases. (B) Abundance of sequences with distinct energy contributions to the kissing complex stability. Red arrows indicate positions of chosen sequences within the energy spectrum. Note that only a few sequences are very stable or very unstable due to the small number of possible CG or AU combinations.

Figure 2

(A) Superimposed melting curves of St1CCGACC and St2GGUCGG complexes. The absorption versus temperature plots for two heating and two cooling runs are displayed. (B) Superimposed first derivatives of the melting curves of St1CCGACC and St2GGUCGG at different RNA concentrations indicated in the indented plot. (C) Van't Hoff analysis of melting curves of hairpins St1CCGACC and St2GGUCGG.

Figure 3

Superimposed first derivatives of melting curves of the kissing complex St1CCGACC/St2GGUCGG at different magnesium concentrations.

Figure 4

Influence of the ionic strength on the melting temperature of the kissing complex St1CCGACC/St2GGUCGG and of a regular RNA helix of identical sequence with dangling adenines. Left curves in black and green indicate the magnesium dependence of the kissing complex and the regular duplex, and the right curves in red and blue indicate sodium dependence of the kissing complex and the regular duplex, respectively. Duplexes at low magnesium concentrations (which always contain 18 mM sodium ions) are observed with T_m values below those for low sodium concentrations. A similar effect has been previously noted (37).