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首页> 外文学位 >The recognition and folding of NCp7 (nucleocapsid protein) with TARDNA and Psi site RNA hairpin structures from HIV1: A spin label study.
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The recognition and folding of NCp7 (nucleocapsid protein) with TARDNA and Psi site RNA hairpin structures from HIV1: A spin label study.

机译：带有HIV1的TARDNA和Psi位点RNA发夹结构的NCp7（核衣壳蛋白）的识别和折叠：自旋标记研究。

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摘要

The nucleocapsid (NC) protein NCp7 of the human immunodeficiency virus type 1 (HIV 1) is a small basic protein with two zinc finger motifs. NCp7 has key roles in virus replication and structure, which rely on its interactions with nucleic acids. We investigated the interaction of NCp7 with Transactivation Response Element (TAR) DNA which is critical for minus strand transfer during the HIV 1 reverse transcription. The EPR spin probe nitroxide was used as the reporter. Binding of NCp7 to TARDNA caused TARDNA condensation, as inferred from the probe tumbling time which markedly increased to several nanoseconds at a 4:1 NCp7 to TARDNA ratio, signifying a NCp7/TARDNA complex with restricted motion. To further investigate NCp7-mediated annealing of TARDNA to the complementary sequence TARRNA, a process which is important for virus replication, Double Electron-Electron Resonance (DEER) was used to directly monitor conformational changes in the TARDNA structure upon NCp7 binding. We found that in the absence of the acceptor TARRNA, NCp7 had only a limited inhibitory effect on the hairpin structure destabilization. In contrast, when both TARDNA and TARRNA were present, NCp7 facilitated formation of the transfer duplex product, and the destabilization of hairpin structure greatly increased. A systematic study of the annealing process of TARDNA with TARRNA hairpins in the presence of NCp7 was performed. These measurements helped to explain why acceptor TARRNA is required for significant annealing of TARDNA by NCp7, and supported the hypothesis that NCp7-mediated annealing of nucleic acids is a concerted process wherein the unfolding step occurs in synchrony with hybridization of complementary TARDNA and TARRNA. Taken as a whole, these studies demonstrated that the spin labeling and DEER measurement provide atomic resolution information of structure and binding interactions.;Stopped-flow EPR kinetics with NCp7/RNASL3 mixed at a 4:1 ratio showed that the interaction of RNASL3 and NCp7 has a concomitant kinetics of probe immobilization from milliseconds to seconds. This work points the way for spin-labeling to investigate oligonucleotide-protein complexes, notably those lacking precise stoichiometry, that are requisite for HIV 1 viral packaging and genome fabrication.
机译：1型人类免疫缺陷病毒（HIV 1）的核衣壳（NC）蛋白NCp7是一种小的碱性蛋白，带有两个锌指基序。 NCp7在病毒复制和结构中起关键作用，这取决于其与核酸的相互作用。我们调查了NCp7与反式激活元件（TAR）DNA的相互作用，这对于HIV 1逆转录过程中负链转移至关重要。 EPR自旋探针氮氧化物用作报告基因。 NCp7与TARDNA的结合导致TARDNA缩合，这是由于探针的翻滚时间推断的，该时间在NCp7与TARDNA的比例为4：1时明显增加到几纳秒，表明NCp7 / TARDNA复合物运动受限。为了进一步研究NCp7介导的TARDNA与互补序列TARRNA的退火，这是病毒复制的重要过程，使用双电子-电子共振（DEER）直接监测NCp7结合后TARDNA结构的构象变化。我们发现在没有受体TARRNA的情况下，NCp7对发夹结构失稳的抑制作用有限。相反，当同时存在TARDNA和TARRNA时，NCp7促进转移双链体产物的形成，发夹结构的不稳定大大增加。在存在NCp7的情况下，对TARDNA与TARRNA发夹的退火过程进行了系统研究。这些测量结果有助于解释为什么NCp7进行TARDNA的显着退火为什么需要受体TARRNA，并支持了NCp7介导的核酸退火是协同过程的假设，其中展开步骤与互补TARDNA和TARRNA的杂交同步发生。总体而言，这些研究表明自旋标记和DEER测量提供了结构和结合相互作用的原子分辨率信息。;以4：1的比例混合NCp7 / RNASL3的停流EPR动力学表明，RNASL3和NCp7的相互作用具有从几毫秒到几秒的固定探针的动力学。这项工作为自旋标记研究寡核苷酸-蛋白质复合物（特别是缺乏精确化学计量的复合物）指明了方向，这是HIV 1病毒包装和基因组制造所必需的。

著录项

  • 作者

    Sun, Yan.;

  • 作者单位

    State University of New York at Albany.;

  • 授予单位 State University of New York at Albany.;
  • 学科 Chemistry Biochemistry.
  • 学位 Ph.D.
  • 年度 2009
  • 页码 170 p.
  • 总页数 170
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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