Non-independence of Mnt repressor-operator interaction determined by a new quantitative multiple fluorescence relative affinity (QuMFRA) assay
Non-independence of Mnt repressor-operator interaction determined by a new quantitative multiple fluorescence relative affinity (QuMFRA) assay
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DOI：
10.1093/nar/29.12.2471
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发表时间：
2001-06-15
影响因子：
14.9
通讯作者：
Stormo, GD
中科院分区：
文献类型：
--
作者：
Man, TK;Stormo, GD
沙门氏菌噬菌体抑制因子Mnt属于带状-螺旋-螺旋转录因子类。以前的SELEX结果表明，MNT与操纵子DNA的16和17位的相互作用不是独立的。利用一种新发展的高通量定量多重荧光相对亲和力(QuMFRA)分析，我们直接定量了MNT与在这两个位置上携带所有可能的二核苷酸组合的操纵子的相对平衡结合常数(K-ref)。结果表明，当17位野生型C改变为其他碱基时，Mnt更倾向于与C结合，而不是野生型A。双突变体的K-ref测定值也高于单突变体的预测值，说明这两个位置的非独立性。同时产生大量定量结合数据的能力和放大的潜力使QuMFRA成为大规模研究大分子相互作用的宝贵工具。
Salmonella bacteriophage repressor Mnt belongs to the ribbon-helix-helix class of transcription factors. Previous SELEX results suggested that interactions of Mnt with positions 16 and 17 of the operator DNA are not independent. Using a newly developed high-throughput quantitative multiple fluorescence relative affinity (QuMFRA) assay, we directly quantified the relative equilibrium binding constants (K-ref) of Mnt to operators carrying all the possible dinucleotide combinations at these two positions. Results show that Mnt prefers binding to C, instead of wildtype A, at position 16 when wild-type C at position 17 is changed to other bases. The measured K-ref values of double mutants were also higher than the values predicted from single mutants, demonstrating the non-independence of these two positions. The ability to produce a large number of quantitative binding data simultaneously and the potential to scale up makes QuMFRA a valuable tool for the large-scale study of macromolecular interaction.