Design of Synthetic Combinatorial Transcription Factors

描述(申请人提供)：许多生物决定由复杂的转录回路控制。能够专门调节这种回路的试剂将非常有用，可以作为解剖复杂过程的顺式调控逻辑的工具，最终作为治疗学。最好的工具是合成的DNA结合蛋白，但这些缺乏在自然转录因子中观察到的组合特异性。最终，在一系列设计师因素的共同作用下，可以实现更精确的监管。许多天然的组合转录因子充当变构开关--该因子只有在另一个辅助因子存在的情况下才有效。最近，一种新的策略被开发出来，用于引入变构控制蛋白质的活性。这一策略包括在蛋白质中引入构象自抑制相互作用；自我抑制可以通过与适当的伙伴蛋白结合来缓解。我的具体目标是使用这种自我抑制策略来产生合成转录因子，其中：1)DNA结合活性由相邻的DNA结合辅助因子变构调节。2)转录激活功能受相邻DNA结合辅助因子的变构调节。这些设计者因素应该产生极其严格的转录控制--激活依赖于分子的精确组合的同时存在。

DESCRIPTION (provided by applicant): Many biological decisions are controlled by complex transcriptional circuits. Reagents that can specifically modulate such circuits would be extremely useful, as tools for dissecting the cis-regulatory logic of complex processes, and ultimately as therapeutics. The best developed tools are synthetic DNA binding proteins, but these lack the combinatorial specificity observed in natural transcription factors. Ultimately, more precise regulation could be achieved with sets of designer factors that act in concert. Many natural combinatorial transcription factors act as allosteric switches -- the factor is only active in the presence of another co-factor. Recently a novel strategy for introducing allosteric control over a protein activity has been developed. This strategy involves the introduction of conformational autoinhibitory interactions into a protein; lautoinhibition can be relieved by association with an appropriate partner protein. My specific aims are to use Ithis autoinhibitory strategy to generate synthetic transcription factors in which: 1) DNA binding activity is allosterically regulated by an adjacent DNA binding co-factor. 2) Transcriptional activation function is allosterically regulated by an adjacent DNA binding co-factor. These designer factors should yield extremely tight transcriptional control -- activation is dependent on the simultaneous presence of a precise combination of molecules.