Department of Integrative Structural and Computational Biology and the Skaggs Institute for Chemical Biology, The Scripps Research Institute , La Jolla, California 92037, United States.
Biochemistry. 2014 Mar 11;53(9):1495-510. doi: 10.1021/bi401675h. Epub 2014 Feb 27.
The interactions of the human double-stranded RNA-binding zinc finger protein JAZ with RNA or DNA were investigated using electrophoretic mobility-shift assays, isothermal calorimetry, and nuclear magnetic resonance spectroscopy. Consistent with previous reports, JAZ has very low affinity for duplex DNA or single-stranded RNA, but it binds preferentially to double-stranded RNA (dsRNA) with no detectable sequence specificity. The affinity of JAZ for dsRNA is unaffected by local structural features such as loops, overhangs, and bulges, provided a sufficient length of reasonably well-structured A-form RNA (about 18 bp for a single zinc finger) is present. Full-length JAZ contains four Cys2His2 zinc fingers (ZF1-4) and has the highest apparent affinity for dsRNA; two-finger constructs ZF12 and ZF23 have lower affinity, and ZF34 binds even more weakly. The fourth zinc finger, ZF4, has no measurable RNA-binding affinity. Single zinc finger constructs ZF1, ZF2, and ZF3 show evidence for multiple-site binding on the minimal RNA. Fitting of quantitative NMR titration and isothermal calorimetry data to a two-site binding model gave Kd1 ∼ 10 μM and Kd2 ∼ 100 μM. Models of JAZ-RNA complexes were generated using the high-ambiguity-driven biomolecular docking (HADDOCK) program. Single zinc fingers bind to the RNA backbone without sequence specificity, forming complexes with contacts between the RNA minor groove and residues in the N-terminal β strands and between the major groove and residues in the helix-kink-helix motif. We propose that the non-sequence-specific interaction between the zinc fingers of JAZ with dsRNA is dependent only on the overall shape of the A-form RNA.
使用电泳迁移率变动分析、等温滴定量热法和核磁共振波谱法研究了人双链 RNA 结合锌指蛋白 JAZ 与 RNA 或 DNA 的相互作用。与先前的报道一致，JAZ 与双链 DNA 或单链 RNA 的亲和力非常低，但它优先与双链 RNA（dsRNA）结合，且没有可检测到的序列特异性。JAZ 与 dsRNA 的亲和力不受局部结构特征（如环、突出和凸起）的影响，只要存在足够长度的结构合理的 A 型 RNA（单个锌指约 18 个碱基）即可。全长 JAZ 包含四个 Cys2His2 锌指（ZF1-4），对 dsRNA 的表观亲和力最高；双锌指结构 ZF12 和 ZF23 的亲和力较低，ZF34 的结合力更弱。第四个锌指 ZF4 没有可测量的 RNA 结合亲和力。单个锌指结构 ZF1、ZF2 和 ZF3 显示出在最小 RNA 上具有多位点结合的证据。对定量 NMR 滴定和等温滴定量热法数据进行拟合，得到 Kd1∼10 μM 和 Kd2∼100 μM。使用高不确定性驱动的生物分子对接（HADDOCK）程序生成 JAZ-RNA 复合物模型。单个锌指与 RNA 骨架无序列特异性结合，形成复合物时，RNA 小沟与 N 端β链上的残基以及大沟与螺旋-环-螺旋模体中的残基之间存在接触。我们提出，JAZ 的 dsRNA 非序列特异性相互作用仅依赖于 A 型 RNA 的整体形状。
