Influence of Bulky Polynuclear Carcinogen Lesions in a TATA Promoter Sequence on TATA Binding Protein-DNA Complex Formation
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- 作者：Olga Rechkoblit ; Jacek Krzeminsky ; Shantu Amin ; Bengt Jernströ ; m ; Natalia Louneva ; and Nicholas E. Geacintov
- 刊名：Biochemistry
- 出版年：2001
- 出版时间：May 15, 2001
- 年：2001
- 卷：40
- 期：19
- 页码：5622 - 5632
- 全文大小：533K
- 年卷期：v.40,no.19(May 15, 2001)
- ISSN：1520-4995
文摘
The TATA binding protein (TBP) is an essential component of the transcription initiationcomplex 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, positionedsite-specifically within a regulatory TATA DNA sequence, on the binding of TBP. Two 25-meroligonucleotides with stereoisomeric 10S (+)-trans-anti- or 10R (-)-trans-anti-BPDE-N6-dA residues atA1 or A2 within the TATA sequence element (5'-...TA1TAAA...-3')·(5'-...TTTA2TA...) were synthesized(anti-BPDE-N6-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 differentTBP concentrations (0-70 nM). The overall affinity of TBP for the BPDE-modified target DNA sequenceswas weakly enhanced in the case of the (+)-trans or (-)-trans lesions positioned at site A1 with Kd 
8and 6 nM, respectively (Kd 9 nM for the unmodified TATA DNA). Higher-order TBP-DNA complexeswere observed at TBP concentrations in excess of ~15 nM. However, the stabilities of the biologicallysignificant monomeric TBP-DNA complexes was dramatically increased or decreased, depending onthe position of the lesion (A1 or A2), or on its stereochemical and conformational characteristics. A moleculardocking modeling approach was employed to insert the stereoisomeric BPDE residues into the knownTATA box-TBP structure [Nikolov, D. B., et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 4862-4867] torationalize these observations. Native gel electrophoresis experiments with the same duplexes withoutTBP indicate that none of the modified sequences exhibit unusual bending induced by the lesions, northat they differ from one another in this respect. These results suggest that the hydrophobic, bulky BPDEresidues influence the binding of TBP by mechanisms other than prebending. The efficiency of RNAtranscription of TBP-controlled promoters could be strongly influenced by the presence of such bulkylesions that could adversely affect the levels of gene expression.
8and 6 nM, respectively (Kd 9 nM for the unmodified TATA DNA). Higher-order TBP-DNA complexeswere observed at TBP concentrations in excess of ~15 nM. However, the stabilities of the biologicallysignificant monomeric TBP-DNA complexes was dramatically increased or decreased, depending onthe position of the lesion (A1 or A2), or on its stereochemical and conformational characteristics. A moleculardocking modeling approach was employed to insert the stereoisomeric BPDE residues into the knownTATA box-TBP structure [Nikolov, D. B., et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 4862-4867] torationalize these observations. Native gel electrophoresis experiments with the same duplexes withoutTBP indicate that none of the modified sequences exhibit unusual bending induced by the lesions, northat they differ from one another in this respect. These results suggest that the hydrophobic, bulky BPDEresidues influence the binding of TBP by mechanisms other than prebending. The efficiency of RNAtranscription of TBP-controlled promoters could be strongly influenced by the presence of such bulkylesions that could adversely affect the levels of gene expression.