Published online 30 September 2004
Nucleic Acids Research, Vol. 32 No. 17 © Oxford University Press 2004; all rights reserved
Determination of thermodynamic parameters for HIV DIS type loop–loop kissing complexes
Max F. Perutz Laboratories, Department of Microbiology and Genetics, University of Vienna, Austria, 1 Institute of Molecular and Cellular Biology of the C.N.R.S., UPR9002, University Louis Pasteur, Strasbourg, France and 2 Department of Theoretical Chemistry and Structural Biology, University of Vienna, Austria
* To whom correspondence should be addressed. Tel: +43 1 4277 54690; Fax: +43 1 4277 9546; Email: renee.schroeder{at}univie.ac.at
Received as resubmission August 1, 2004; Revised and Accepted September 3, 2004
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 Tm/log [ion] plot. The short base-paired helices are stabilized by 8°C/log [Mg2+] or 11°C/log [Na+], whereas base-paired helices forming tertiary loop–loop interactions are stabilized by 16°C/log [Mg2+] and 26°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.
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