Neely L S, Lee B M, Xu J, Wright P E, Gottesfeld J M
Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
J Mol Biol. 1999 Aug 20;291(3):549-60. doi: 10.1006/jmbi.1999.2985.
Transcription factor IIIA of Xenopuslaevis serves a dual function during oogenesis and early development: this zinc finger protein binds to the internal promoter element of the 5 S ribosomal RNA genes and acts as a positive transcription factor; additionally, the protein functions in 5 S RNA storage. The central four zinc fingers (zf4-7) of the nine-finger protein have been shown to bind 5 S rRNA with comparable or higher affinity than the full-length protein. The role of finger seven in binding affinity has been examined by deletion analysis. A zf4-6 protein binds 5 S RNA with about a sevenfold reduction in binding affinity, compared to zf4-7. The effect of non-specific competitor DNA on binding affinities of the zinc finger peptides was examined and found to have a significant effect on the measured affinities of these peptides for full-length and truncated versions of 5 S RNA. The interaction of zf4-6 with full-length 5 S RNA was far more sensitive to non-specific competitor concentration than was the zf4-7:5 S RNA interaction, suggesting that finger seven contributes to both affinity and specificity in this protein:RNA interaction. In order to map zinc finger binding sites on the 5 S RNA molecule, we generated truncated versions of the RNA and tested these molecules for their binding affinities with zf4-7 and zf4-6. Previous studies showed that a 75 nucleotide long RNA, comprising loop A, helix II, helix V, region E and helix IV, bound zf4-7 with high affinity. Selection and amplification binding assays (selex) have now been used to generate smaller high-affinity binding RNAs. We find that a 55 nucleotide long RNA, comprising loop A, helix V, region E and helix IV, but lacking helix II, retains high affinity for zf4-6. These data are consistent with the proposal that fingers 4-6 bind this central core of 5 S RNA and that finger seven binds the helix II region.
非洲爪蟾的转录因子IIIA在卵子发生和早期发育过程中发挥双重功能：这种锌指蛋白与5S核糖体RNA基因的内部启动子元件结合，并作为正转录因子发挥作用；此外，该蛋白在5S RNA储存中起作用。已证明九指蛋白的中央四个锌指（zf4 - 7）与5S rRNA结合的亲和力与全长蛋白相当或更高。通过缺失分析研究了锌指七在结合亲和力中的作用。与zf4 - 7相比，zf4 - 6蛋白与5S RNA结合的亲和力降低了约七倍。研究了非特异性竞争DNA对锌指肽结合亲和力的影响，发现其对这些肽与5S RNA全长和截短版本的测量亲和力有显著影响。zf4 - 6与全长5S RNA的相互作用对非特异性竞争剂浓度的敏感性远高于zf4 - 7与5S RNA的相互作用，这表明锌指七在这种蛋白质与RNA的相互作用中对亲和力和特异性都有贡献。为了绘制锌指在5S RNA分子上的结合位点，我们生成了RNA的截短版本，并测试了这些分子与zf4 - 7和zf4 - 6的结合亲和力。先前的研究表明，一个由环A、螺旋II、螺旋V、区域E和螺旋IV组成且长度为75个核苷酸的RNA与zf4 - 7具有高亲和力。现在已使用选择和扩增结合测定（selex）来生成更小的高亲和力结合RNA。我们发现一个由环A、螺旋V、区域E和螺旋IV组成但缺少螺旋II且长度为55个核苷酸的RNA对zf4 - 6仍保持高亲和力。这些数据与以下提议一致，即锌指4 - 6结合5S RNA的这个中央核心，而锌指七结合螺旋II区域。
