Laboratory of Molecular Genetics, Centre for DNA Fingerprinting and Diagnostics, Nampally, Hyderabad 500001, India.
Nucleic Acids Res. 2011 Dec;39(22):9574-91. doi: 10.1093/nar/gkr672. Epub 2011 Sep 2.
Many proteins of the Rel family can act as both transcriptional activators and repressors. However, mechanism that discerns the 'activator/repressor' functions of Rel-proteins such as Dorsal (Drosophila homologue of mammalian NFκB) is not understood. Using genomic, biophysical and biochemical approaches, we demonstrate that the underlying principle of this functional specificity lies in the 'sequence-encoded structure' of the κB-DNA. We show that Dorsal-binding motifs exist in distinct activator and repressor conformations. Molecular dynamics of DNA-Dorsal complexes revealed that repressor κB-motifs typically have A-tract and flexible conformation that facilitates interaction with co-repressors. Deformable structure of repressor motifs, is due to changes in the hydrogen bonding in A:T pair in the 'A-tract' core. The sixth nucleotide in the nonameric κB-motif, 'A' (A(6)) in the repressor motifs and 'T' (T(6)) in the activator motifs, is critical to confer this functional specificity as A(6) → T(6) mutation transformed flexible repressor conformation into a rigid activator conformation. These results highlight that 'sequence encoded κB DNA-geometry' regulates gene expression by exerting allosteric effect on binding of Rel proteins which in turn regulates interaction with co-regulators. Further, we identified and characterized putative repressor motifs in Dl-target genes, which can potentially aid in functional annotation of Dorsal gene regulatory network.
许多 Rel 家族的蛋白既可以作为转录激活剂，也可以作为转录抑制剂。然而，区分 Dorsal（哺乳动物 NFκB 的果蝇同源物）等 Rel 蛋白的“激活剂/抑制剂”功能的机制尚不清楚。我们使用基因组、生物物理和生化方法证明，这种功能特异性的基本原则在于κB-DNA 的“序列编码结构”。我们表明 Dorsal 结合基序存在于不同的激活剂和抑制剂构象中。DNA-Dorsal 复合物的分子动力学研究表明，抑制剂κB 基序通常具有 A 链和柔性构象，这有利于与共抑制剂相互作用。抑制剂基序的可变形结构是由于“A 链”核心中 A:T 对氢键的变化所致。非环六核苷酸κB 基序中的第六个核苷酸，抑制剂基序中的“A”（A(6)) 和激活剂基序中的“T”（T(6))，对于赋予这种功能特异性至关重要，因为 A(6)→T(6) 突变将柔性抑制剂构象转化为刚性激活剂构象。这些结果表明，“序列编码的 κB DNA 几何形状”通过对 Rel 蛋白结合施加变构效应来调节基因表达，从而调节与共调节剂的相互作用。此外，我们鉴定并表征了 Dl 靶基因中的假定抑制剂基序，这可能有助于 Dorsal 基因调控网络的功能注释。
