Conformational Characteristics of High Affinity Sp1 Binding Enhancer Elements of HIV-LTR by High Resolution 2D-NMR

Abstract

The understanding of early events in the expression of genes has vastly improved in recent years with the identification of a variety of gene- and sequence-specific DNA binding transcription factors. One such protein, Sp1, has been implicated in activating transcription of various cellular and viral genes including those of HIV, and SIV types of retroviruses. The basic recognition site for Sp1 has been identified as variants of a 10 base-pairs long GC-rich DNA, often containing a hexanucleotide segment 5'-GGGCGG (termed GC-box). However, variations in both the relative protein-DNA binding affinity and the nature of binding sequences have been noted. Two-dimensional 1H-NMR experiments (500 MHz) were employed for conformational studies of two decadeoxyribonucleotide duplexes, d(GAGGCGTGGC).d(GCCACGCCTC), termed Sp1-III, and d(GGGAGTGGCG).d(CGCCACTCCC), termed Sp1-I. These are two of the highest affinity Sp1 binding sites and consist of diverse positioning of the tri- and tetranucleotide segments GAG, GTG, GCG, GGCG, GTGG and GGAG, that occur frequently in other Sp1 binding sites as well, and may form specific contacts with the protein. Phase-sensitive nuclear Overhauser enhancement (2D-NOESY and MINSY) and correlation (COSY) spectra were obtained for the assignment of the exchangeable and nonexchangeable protons in a sequence-specific fashion. As a prelude to determination of the detailed solution structures of the selected sequences, numerous structural constraints were obtained from angle-dependent coupling constants and relative intensities of distance-dependent intra- and internucleotide NOEs. Overall, each duplex adopts a structure similar to B-DNA with predominantly C2'-endo/S-type sugar conformation and anti-glycosidic torsion angles. A selective disruption of sequential NOE connectivities at the GAG.CAC and GTG.CAC steps, irrespective of the flanking sequence, suggests that conformational changes at these sites may act as unique determinants of sequence specific recognition/binding of Sp1. Implications for a specific inhibition of Sp1-mediated transcription by minor groove binding class of drugs, designed to recognize GC-rich sequences, are also briefly discussed.