Finger-positional change in three zinc finger protein Sp1: influence of terminal finger in DNA recognition.
Finger-positional change in three zinc finger protein Sp1: influence of terminal finger in DNA recognition.
复制标题
三个锌指蛋白 Sp1 的指位置变化：末端指对 DNA 识别的影响。
作者：
Y. Uno;K. Matsushita;M. Nagaoka;Y. Sugiura
功能模块的连接对于设计具有所需序列特异性的DNA结合分子是有效的。C(2)H(2)型锌指蛋白具有串联重复的阵列结构，由独立的指模组成，有望通过排列、多连接和各种锌指组的替换来识别任何DNA序列。为了研究末端指的替换对其他指的DNA识别的影响，我们构建了在N端或c端进行指替换的锌指肽Sp1（zf223）、Sp1（zf323）和Sp1（zf321）。从凝胶迁移位移测定的结果来看，如果手指以模块化的方式起作用，每个突变肽优先结合到预测的目标序列。甲基化干扰分析表明，在n端手指替换突变体Sp1（zf223）和Sp1（zf323）的情况下，n端手指识别碱基的程度与野生型肽Sp1（zf123）不同。特别有趣的是，c端手指替换突变体Sp1（zf321）的n端手指与Sp1（zf123）和Sp1（zf323）的碱基识别明显不同。DNA酶I足迹分析表明，c端指（主动指）在结合n端指（被动指）的区域诱导DNA构象变化。目前的结果强烈表明，n端手指的碱基识别程度主要是由c端手指的结合决定的。这一信息为创造具有所需特异性的锌指肽提供了重要线索，适用于新药和生物工具的设计。
The connection of functional modules is effective for the design of DNA binding molecules with the desired sequence specificity. C(2)H(2)-type zinc finger proteins have a tandemly repeated array structure consisting of independent finger modules and are expected to recognize any DNA sequences by permutation, multi-connection, and the substitution of various sets of zinc fingers. To investigate the effects of the replacement of the terminal finger on the DNA recognition by other fingers, we have constructed the three zinc finger peptides with finger substitution at the N- or C-terminus, Sp1(zf223), Sp1(zf323), and Sp1(zf321). From the results of gel mobility shift assays, each mutant peptide binds preferentially to the target sequence that is predicted if the fingers act in a modular fashion. The methylation interference analyses demonstrate that in the cases of the N-terminal finger substitution mutants, Sp1(zf223) and Sp1(zf323), the N-terminal finger recognizes bases to different extents from that of the wild-type peptide, Sp1(zf123). Of special interest is the fact that the N-terminal finger of the C-terminal finger substitution mutant, Sp1(zf321), shows a distinct base recognition from those of Sp1(zf123) and Sp1(zf323). DNase I footprinting analyses indicate that the C-terminal finger (active finger) induces a conformational change in the DNA in the region for the binding of the N-terminal finger (passive finger). The present results strongly suggest that the extent of base recognition of the N-terminal finger is dominated by the binding of the C-terminal finger. This information provides an important clue for the creation of a zinc finger peptide with the desired specificity, which is applicable to the design of novel drugs and biological tools.