Park C, Campbell J L, Goddard W A
Materials and Molecular Simulation Center, Beckman Institute (139-74), Pasadena, CA.
Proc Natl Acad Sci U S A. 1992 Oct 1;89(19):9094-6. doi: 10.1073/pnas.89.19.9094.
We present a general strategy for designing proteins to recognize DNA sequences and illustrate this with an example based on the "Y-shaped scissors grip" model for leucine-zipper gene-regulatory proteins. The designed protein is formed from two copies, in tandem, of the basic (DNA binding) region of v-Jun. These copies are coupled through a tripeptide to yield a "dimer" expected to recognize the sequence TCATCGATGA (the v-Jun-v-Jun homodimer recognizes ATGACTCAT). We synthesized the protein and oligonucleotides containing the proposed binding sites and used gel-retardation assays and DNase I footprinting to establish that the dimer binds specifically to the DNA sequence TCATCGATGA but does not bind to the wild-type DNA sequences, nor to oligonucleotides in which the recognition half-site is modified by single-base changes. These results also provide strong support for the Y-shaped scissors grip model for binding of leucine-zipper proteins.
我们提出了一种设计能识别DNA序列的蛋白质的通用策略，并以基于亮氨酸拉链基因调控蛋白的“Y形剪刀夹”模型的一个例子进行说明。设计的蛋白质由v-Jun基本（DNA结合）区域的两个串联拷贝组成。这些拷贝通过一个三肽连接，产生一个预期能识别序列TCATCGATGA的“二聚体”（v-Jun-v-Jun同二聚体识别ATGACTCAT）。我们合成了该蛋白质和含有拟议结合位点的寡核苷酸，并使用凝胶阻滞试验和DNase I足迹法来确定二聚体特异性结合DNA序列TCATCGATGA，但不结合野生型DNA序列，也不结合识别半位点经单碱基改变修饰的寡核苷酸。这些结果也为亮氨酸拉链蛋白结合的Y形剪刀夹模型提供了有力支持。
