Smith J F, Hawkins J, Leonard R E, Hanas J S
Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City 73190.
Nucleic Acids Res. 1991 Dec 25;19(24):6871-6. doi: 10.1093/nar/19.24.6871.
Zinc binding domains and the conserved Thr-Gly-Glu-Lys (TGEK) tetrapeptide in the N-terminal half of transcription factor IIIA (TFIIIA) were subjected to in vitro mutagenesis to biochemically assess their role in factor interaction with the 5S gene internal control region (ICR). TFIIIA containing a Leu in place of His33 in the Cys2His2 zinc binding site of finger I lost the ability to protect the entire 5S RNA gene ICR (nucleotides +96 to +43) from DNase I digestion. Thus, mutation of one potential zinc ligand in the N-terminal finger inhibited specific DNA binding by the N-terminal as well as downstream fingers. Cooperativity apparently exists among TFIIIA zinc fingers in metal binding/finger folding and DNA binding. Substituting a Ser for Gly69 or a Glu for Lys 71 in the conserved TGEK tetrapeptide in finger II of TFIIIA resulted in the loss of DNA binding. A Gly-dependent bend structure and a terminal positive charge in this tetrapeptide are important for TFIIIA interaction with DNA. Whereas TFIIIA with a Ser substituted for Cys20 in finger I (proposed zinc ligand) did not protect the ICR from DNase I digestion, TFIIIA containing a Ser substituted for Cys35 (not a proposed zinc ligand) retained the ability to bind the ICR. When Cys112 or Cys 164 (proposed zinc ligands in fingers IV and VI) were replaced by Ser, the DNase I footprint patterns afforded by the respective mutant proteins were similar, protection on the ICR from about nucleotides +96 up to +78. A similar pattern was obtained with a TFIIIA mutant in which fingers V, VI, VII, and a portion of VIII were deleted. Maintenance of zinc coordination spheres in necessary for DNA binding by downstream fingers. The six fingers comprising the N-terminal half of TFIIIA appear to act in two groups of three with binding of the second group dependent upon initial binding of the N-terminal group to the +90 to +80 region of the 5S gene ICR.
对转录因子IIIA（TFIIIA）N端的锌结合结构域以及保守的苏氨酸 - 甘氨酸 - 谷氨酸 - 赖氨酸（TGEK）四肽进行体外诱变，以生物化学方式评估它们在该因子与5S基因内部控制区（ICR）相互作用中的作用。在手指I的Cys2His2锌结合位点中，用亮氨酸取代His33的TFIIIA失去了保护整个5S RNA基因ICR（核苷酸+96至+43）免受DNase I消化的能力。因此，N端手指中一个潜在锌配体的突变抑制了N端以及下游手指的特异性DNA结合。TFIIIA锌指在金属结合/手指折叠和DNA结合方面显然存在协同作用。在TFIIIA手指II的保守TGEK四肽中，用丝氨酸取代Gly69或用谷氨酸取代Lys 71会导致DNA结合丧失。该四肽中依赖甘氨酸的弯曲结构和末端正电荷对于TFIIIA与DNA的相互作用很重要。虽然在手指I中用丝氨酸取代Cys20（推测的锌配体）的TFIIIA不能保护ICR免受DNase I消化，但含有丝氨酸取代Cys35（不是推测的锌配体）的TFIIIA保留了结合ICR的能力。当Cys112或Cys 164（手指IV和VI中推测的锌配体）被丝氨酸取代时，各自突变蛋白提供的DNase I足迹模式相似，对ICR从大约核苷酸+96到+78有保护作用。在一个TFIIIA突变体中也获得了类似的模式，其中手指V、VI、VII和VIII的一部分被删除。维持锌配位球对于下游手指的DNA结合是必要的。构成TFIIIA N端一半的六个手指似乎以两组三个的形式起作用，第二组的结合取决于N端组与5S基因ICR的+90至+80区域的初始结合。
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