Influence of bulky polynuclear carcinogen lesions in a TATA promoter sequence on TATA binding protein-DNA complex formation

The TATA binding protein (TBP) is an essential component of the transcripti on initiation complex that recognizes and binds to the minor groove of the TATA DNA duplex consensus sequences. The objective of this study was to det ermine 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 sequen ce element (5'-...TA(1)TAAA...-3').(5'-...TTTA(2)TA...) were synthesized (a nti-BPDE-N-6-dA denotes an adduct formed from the reaction of r7,t8-dihydro xy-t9, 10-epoxy-7,8,9,-10-tetrahydobenzo[a]pyrene). The formation of comple xes with TBP of these two sequences in the double-stranded forms (1 nM) wer e studied employing electrophoretic mobility shift assays (EMSA) at differe nt TBP concentrations (0-70 nM), The overall affinity of TBP for the BPDE-m odified target DNA sequences was weakly enhanced in the case of the (+)-tra ns or (-)-trans lesions positioned at site A(1) with K-d approximate to 8 a nd 6 nM, respectively (K-d approximate to 9 nM for the unmodified TATA DNA) . Higher-order TBP-DNA complexes were observed at TBP concentrations in exc ess of similar to 15 nM. However, the stabilities of the biologically signi ficant monomeric TBP-DNA complexes was dramatically increased or decreased, depending on the position of the lesion (A(1) or A(2)), Or On its stereoch emical and conformational characteristics. A molecular docking modeling app roach was employed to insert the stereoisomeric BPDE residues into the know n TATA box-TBP structure [Nikolov, D. B., et al. (1996) Proc. Natl. Acad. S ci. U.S.A. 4862-4867] to rationalize these observations. Native gel electro phoresis experiments with the same duplexes without TBP indicate that none of the modified sequences exhibit unusual bending induced by the lesions, n or 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 TB P-controlled promoters could be strongly influenced by the presence of such bulky lesions that could adversely affect the levels of gene expression.