Hanas J S, Koelsch G, Moreland R, Wickham J Q
Department of Biochemistry and Molecular Biology, University of Oklahoma College of Medicine, 940 Stanton Young Blvd., Oklahoma City, OK 73140, USA.
Biochim Biophys Acta. 1998 Jul 9;1398(3):256-64. doi: 10.1016/s0167-4781(98)00070-0.
Basic amino acids Arg, Lys, and His in the Cys2His2 zinc fingers of transcription factor IIIA (TFIIIA) potentially have important roles in factor binding to the extended internal control region (ICR) of the 5S ribosomal gene. Conserved and non-conserved basic residues in the N-terminal fingers I, II, III and the more C-terminal fingers V and IX were analyzed by site-directed mutagenesis and DNase I protection in order to assess their individual requirement in the DNA-binding mechanism. In the DNA recognition helix of finger II, the conserved Arg at position 62 (N-terminal side of the first zinc-coordinating histidine) was changed to a Leu or Gln. Both the R62L and R62Q mutations inhibited Xenopus TFIIIA-dependent DNase I footprinting along the entire 5S gene ICR. When His-58 (non-conserved basic residue with DNA-binding potential in the same helical region) was changed to a Gln, the mutated protein was able to protect the ICR from DNase I digestion. Therefore, Arg-62 is individually required for TFIIIA binding over the entire ICR whereas His-58 is not. Fingers V and IX have conserved Arg residues in positions identical to Arg-62 in finger II (Arg-154 in finger V and Arg-271 in finger IX). When these residues were changed to Leu and Ile respectively, TFIIIA-dependent DNase I protection was observed along the entire 5S gene ICR. These results indicate differing DNA-binding mechanisms by the N-terminal fingers versus the C-terminal fingers at the level of individual amino acid-nucleotide interactions. In the N-terminal finger I, the conserved Lys at position 11 outside the recognition helix and a conserved hydrophobic Trp at position 28 within the helix were changed to an Ala and Ser respectively. The K11A change inhibited TFIIIA-dependent DNase I protection to a much greater extent than the W28S change.
转录因子IIIA（TFIIIA）的Cys2His2锌指中的碱性氨基酸精氨酸（Arg）、赖氨酸（Lys）和组氨酸（His）可能在该因子与5S核糖体基因的延伸内部控制区（ICR）结合中发挥重要作用。通过定点诱变和DNase I保护分析了N端的I、II、III指以及C端的V和IX指中保守和非保守的碱性残基，以评估它们在DNA结合机制中的各自需求。在指II的DNA识别螺旋中，第62位（第一个锌配位组氨酸的N端侧）的保守精氨酸被替换为亮氨酸（Leu）或谷氨酰胺（Gln）。R62L和R62Q突变均抑制了非洲爪蟾TFIIIA依赖的沿整个5S基因ICR的DNase I足迹分析。当第58位组氨酸（同一螺旋区域中具有DNA结合潜力的非保守碱性残基）被替换为谷氨酰胺时，突变蛋白能够保护ICR免受DNase I消化。因此，精氨酸-62是TFIIIA在整个ICR上结合所单独需要的，而组氨酸-58则不是。V指和IX指在与指II中精氨酸-62相同的位置（V指中的精氨酸-154和IX指中的精氨酸-271）有保守的精氨酸残基。当这些残基分别被替换为亮氨酸和异亮氨酸时，观察到沿整个5S基因ICR的TFIIIA依赖的DNase I保护。这些结果表明，在单个氨基酸-核苷酸相互作用水平上，N端指与C端指的DNA结合机制不同。在N端的I指中，识别螺旋外第11位的保守赖氨酸和螺旋内第28位的保守疏水色氨酸分别被替换为丙氨酸和丝氨酸。K11A突变比W28S突变更显著地抑制了TFIIIA依赖的DNase I保护。
