RESUMO
The TATA binding protein (TBP) is an essential component of the transcription initiation complex that recognizes and binds to the minor groove of the TATA DNA duplex consensus sequences. The objective of this study was to determine the effect of a carcinogen-modified adenine residue, positioned site-specifically within a regulatory TATA DNA sequence, on the binding of TBP. Two 25-mer oligonucleotides with stereoisomeric 10S (+)-trans-anti- or 10R (-)-trans-anti-BPDE-N(6)-dA residues at A(1) or A(2) within the TATA sequence element (5'-...TA(1)TAAA...-3')-(5'-...TTTA(2)TA...) were synthesized (anti-BPDE-N(6)-dA denotes an adduct formed from the reaction of r7,t8-dihydroxy-t9,10-epoxy-7,8,9,10-tetrahydobenzo[a]pyrene). The formation of complexes with TBP of these two sequences in the double-stranded forms (1 nM) were studied employing electrophoretic mobility shift assays (EMSA) at different TBP concentrations (0-70 nM). The overall affinity of TBP for the BPDE-modified target DNA sequences was weakly enhanced in the case of the (+)-trans or (-)-trans lesions positioned at site A(1) with K(d) approximately 8 and 6 nM, respectively (K(d) approximately 9 nM for the unmodified TATA DNA). Higher-order TBP-DNA complexes were observed at TBP concentrations in excess of approximately 15 nM. However, the stabilities of the biologically significant monomeric TBP-DNA complexes was dramatically increased or decreased, depending on the position of the lesion (A(1) or A(2)), or on its stereochemical and conformational characteristics. A molecular docking modeling approach was employed to insert the stereoisomeric BPDE residues into the known TATA box-TBP structure [Nikolov, D. B., et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 4862-4867] to rationalize these observations. Native gel electrophoresis experiments with the same duplexes without TBP indicate that none of the modified sequences exhibit unusual bending induced by the lesions, nor that they differ from one another in this respect. These results suggest that the hydrophobic, bulky BPDE residues influence the binding of TBP by mechanisms other than prebending. The efficiency of RNA transcription of TBP-controlled promoters could be strongly influenced by the presence of such bulky lesions that could adversely affect the levels of gene expression.
