Solution structures and characterization of human immunodeficiency virus Rev responsive element IIB RNA targeting zinc finger proteins.
Solution structures and characterization of human immunodeficiency virus Rev responsive element IIB RNA targeting zinc finger proteins.
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人类免疫缺陷病毒 Rev 反应元件 IIB RNA 靶向锌指蛋白的溶液结构和表征。
DOI：
10.1002/bip.20565
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发表时间：
2006
期刊：
影响因子：
2.9
通讯作者：
Germann,MarkusW
中科院分区：
文献类型：
--
作者：
Mishra,SubrataH;Shelley,ChristopherM;BarrowJr,DoyleJ;Darby,MartynK;Germann,MarkusW
Rev反应元件（RRE）是未剪接的人类免疫缺陷病毒（HIV）RNA的一部分，在感染性HIV病毒体的产生中起关键作用。病毒蛋白Rev与RRE结合并促进mRNA转运至细胞质。抑制Rev-RRE相互作用会破坏病毒的生命周期。使用噬菌体展示方案，产生在高亲和力Rev结合位点特异性结合RREIIB的双锌指蛋白（ZNF）。这些蛋白质被进一步缩短和简化，它们仍然保留了它们的RNA结合亲和力。用核磁共振（NMR）谱测定了ZNF 29及其突变体ZNF 29 G 29 R的溶液结构。两种蛋白质形成具有基本相同结构的C2 H2-型锌指。RNA-蛋白质相互作用进行了定量评价等温滴定量热法，揭示了解离常数（Kd的）在纳摩尔范围内。与RNA的相互作用取决于锌指结构;在EDTA存在下，RNA结合被消除。对于这两种蛋白质，RNA结合由锌指的α螺旋部分介导，并靶向RREIIB-TR的凸起区域。然而，ZNF 29 G29 R表现出比ZNF 29显著更强的与RNA靶的结合;这说明锌指支架的结合可进一步改进。© 2006 Wiley Periodicals，Inc. Biopoly 83：352-364，2006这篇文章最初作为公认的预印本在网上发表。“在线发布”日期对应于预印本。您可以通过向Biopolymers编辑部发送电子邮件（biopolymers@wiley.com）索取预印本的副本
The Rev responsive element (RRE), a part of unspliced human immunodeficiency virus (HIV) RNA, serves a crucial role in the production of infectious HIV virions. The viral protein Rev binds to RRE and facilitates transport of mRNA to the cytoplasm. Inhibition of the Rev–RRE interaction disrupts the viral life cycle. Using a phage display protocol, dual zinc finger proteins (ZNFs) were generated that bind specifically to RREIIB at the high affinity Rev binding site. These proteins were further shortened and simplified, and they still retained their RNA binding affinity. The solution structures of ZNF29 and a mutant, ZNF29G29R, have been determined by nuclear magnetic resonance (NMR) spectroscopy. Both proteins form C2H2‐type zinc fingers with essentially identical structures. RNA protein interactions were evaluated quantitatively by isothermal titration calorimetry, which revealed dissociation constants (Kd's) in the nanomolar range. The interaction with the RNA is dependent upon the zinc finger structure; in the presence of EDTA, RNA binding is abolished. For both proteins, RNA binding is mediated by the α‐helical portion of the zinc fingers and target the bulge region of RREIIB‐TR. However, ZNF29G29R exhibits significantly stronger binding to the RNA target than ZNF29; this illustrates that the binding of the zinc finger scaffold is amenable to further improvements. © 2006 Wiley Periodicals, Inc. Biopoly 83:352–364, 2006This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com