CONFORMATIONAL STUDIES BY H-1-NMR OF THE HIV ENHANCER - THE TRANSCRIPTION FACTORS NF-KAPPA-B AND SP1 BINDING DOMAINS

The solution structures of two DNA non-palindromic duplexes of 24 base pairs and corresponding to the 3' NF-KB-binding site and the adjacent 5' Sp1-binding site of the HIV-1 long terminal repeat (5'LTR) were an alyzed by proton two-dimensional nuclear magnetic resonance spectrosco py. The first sequence GACTTTCCAGGGAGGCGTGGC):d(GCCACGCCTCCCTGGAAAGTCC CC) corresponds to the wild-type LTR duplex (24mer-N), and the second sequence CACTTTCCAGGGAGGCGTGGC):d(GCCACGCCTCCCTGGAAAGTGAGC) (24mer-M) contains a specific mutation (lightface letters) abolishing NF-KB bind ing, Assignment of protons essential for structure determination were obtained and structural features of both duplexes were analyzed. The o verall structure of the duplex is of the B form but several significan t local structural deviations were found. First, from analysis of NOE cross-peak intensities between adenine H2 base protons and sugar H1' p rotons, it was found that the CTC mutation results in widening of the minor groove with presumably narrowing of the major groove, thus impai ring the binding of NF-KB to its responsive element in the HIV LTR. Se cond from chemical shift analysis of H1' sugar protons, some unusual s tructural features were found at the junction between the homopurine:h omopyrimidine stretches, that is, at the junction of the NF-KB and Sp1 sites, consistent with the known synergism between NF-KB and Sp1 func tions in the HIV LTR. The scopes and limitations of DNA fragment studi es of such a size are discussed.