HIGH-RESOLUTION 2D-NMR STUDIES INCLUDING COMPLETE ASSIGNMENTS AND CONFORMATIONAL CHARACTERISTICS OF THE NF-KAPPA-B BINDING ENHANCER ELEMENTOF HIV-LTR

The asymmetrical DNA duplex [5'-d(AAGGGACTITCC)].[5'-d(GGAAAGTCCCT)] h as been studied by one- and two-dimensional NMR techniques. The sequen ce is comprised of the actual 10 base-pair long binding site for the t ranscription factor NF-kappa B in the enhancer sequence of the long te rm repeat (LTR) region of HIV and SIV types of retroviruses associated with the AIDS syndrome. Two additional A . T base-pairs are also incl uded on one end for an added interest in the 12-bp duplex sequence wit h a pseudo dyad-symmetric disposition of the oligopurine and oligopyri midine segments, as it appears in the HIV-1 genome. Phase-sensitive tw o-dimensional spectra (NOESY, POESY, COSY and TOCSY) were obtained at three different temperatures (5, 15 and 25 degrees C) for a complete a ssignment of the non-exchangeable protons by tracing through sequence specific intra- and internucleotide connectivities. 2D-NOESY spectra w ere also acquired in aqueous (90% H2O-D2O) solutions, with two differe nt methods of water signal suppression, to assign the exchangeable pro tons from specific NOE correlations. Adenine H2 protons were assigned by the use of NOE correlations and from T-1 relaxation time measuremen ts. The general spectral features and semi-quantitative interproton di stance estimates indicate a B-DNA type conformation. However, some dis tinctly unusual features associated with the nucleotides at and immedi ately adjacent to both the 5'- and 3'-ends of AAA/TTT and GGG/CCC segm ents were noted. The complete assignments, and the observed characteri stics, will be of significant value in studying the complexes of this transcriptionally active DNA domain with the protein and other rationa lly designed DNA binding agents.