Influence of bulky polynuclear carcinogen lesions in a TATA promoter sequence on TATA binding protein-DNA complex formation.
Influence of bulky polynuclear carcinogen lesions in a TATA promoter sequence on TATA binding protein-DNA complex formation.
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TATA 启动子序列中大量多核致癌物损伤对 TATA 结合蛋白-DNA 复合物形成的影响。
DOI：
10.1021/bi002543r
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发表时间：
2001
期刊：
影响因子：
2.9
通讯作者：
Geacintov,NE
中科院分区：
文献类型：
--
作者：
Rechkoblit,O;Krzeminsky,J;Amin,S;Jernström,B;Louneva,N;Geacintov,NE
TATA 结合蛋白 (TBP) 是转录起始复合物的重要组成部分，可识别并结合 TATA DNA 双链体共有序列的小沟。本研究的目的是确定致癌物修饰的腺嘌呤残基（特定位点位于 TATA DNA 调节序列内）对 TBP 结合的影响。合成了两个 25 聚体寡核苷酸，在 TATA 序列元件 (5'-...TA1TAAA...-3')·(5'-...TTTA2TA...) 内的 A1 或 A2 处具有立体异构 10S(+)-trans-anti- 或 10R(−)-trans-anti-BPDE-N6-dA 残基（anti-BPDE-N6-dA 表示由反应形成的加合物） (7,t8-二羟基-t9,10-环氧-7,8,9,10-四氢苯并[a]芘)。采用电泳迁移率变动分析 (EMSA) 在不同的 TBP 浓度 (0−70 nM) 下研究了这两个序列的双链形式 (1 nM) 与 TBP 的复合物形成。对于位于 A1 位点的 (+)-反式或 (-)-反式损伤，TBP 对 BPDE 修饰的靶 DNA 序列的总体亲和力分别微弱增强，Kd ≈ 8 和 6 nM（对于未修饰的 TATA DNA，Kd ≈ 9 nM）。在 TBP 浓度超过~15 nM 时观察到高阶 TBP−DNA 复合物。然而，具有生物学意义的单体 TBP-DNA 复合物的稳定性显着增加或降低，具体取决于病变的位置（A1 或 A2）或其立体化学和构象特征。采用分子对接建模方法将立体异构 BPDE 残基插入已知的 TATA 盒-TBP 结构中 [Nikolov, D. B., et al. (1996)Proc。国家。阿卡德。科学。 U.S.A.4862−4867] 合理化这些观察结果。使用不含 TBP 的相同双链体进行的天然凝胶电泳实验表明，没有一个修饰序列表现出由损伤引起的异常弯曲，并且它们在这方面也没有彼此不同。这些结果表明，疏水性大的 BPDE 残基通过预弯曲以外的机制影响 TBP 的结合。 TBP 控制的启动子的 RNA 转录效率可能会受到这种巨大病变的强烈影响，从而对基因表达水平产生不利影响。
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-N6-dA residues at A1or A2within the TATA sequence element (5‘-...TA1TAAA...-3‘)·(5‘-...TTTA2TA...) were synthesized (anti-BPDE-N6-dA denotes an adduct formed from the reaction ofr7,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). Theoverallaffinity of TBP for the BPDE-modified target DNA sequences was weakly enhanced in the case of the (+)-trans or (−)-trans lesions positioned at site A1withKd≈ 8 and 6 nM, respectively (Kd≈ 9 nM for the unmodified TATA DNA). Higher-order TBP−DNA complexes were observed at TBP concentrations in excess of ∼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 (A1or A2), 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.