Khan R, Giedroc D P
Department of Biochemistry and Biophysics, Texas A & M University, College Station 77843-2128.
J Biol Chem. 1992 Apr 5;267(10):6689-95.
The nucleocapsid protein (NC) of all animal retroviruses, encoded by the gag gene, is the major structural protein of the core ribonucleoprotein complex, bound to genomic RNA in mature virions. NC is also thought to play one or more accessory roles in reverse transcription. Mature NC (p7NC) from human immunodeficiency virus type 1 (HIV-1) is a 71-amino acid, basic protein which contains two Cys3His Zn(II) retroviral-type zinc finger domains. Herein, we describe the subcloning and expression of HIV-1 NC, denoted NC71, from an inducible phage T7 RNA polymerase promoter in Escherichia coli. Purified NC71 can be reversibly reconstituted with 2 g.at Zn(II) determined by atomic absorption. Ultraviolet circulation dichroism spectroscopy has been used to characterize the complexes between highly purified NC71 and the RNA homopolynucleotide poly(A) and E. coli tRNA(mixed). On poly(A), Zn2 NC71 is characterized by an apparent site size n = 15 +/- 3 nucleotides and high affinity (Kapp = 3 x 10(7) M-1) and moderately cooperative (omega approximately 170 +/- 25) binding. A mixture of E. coli tRNA species (tRNA(mixed) was used to probe the conformational changes induced in tRNA upon binding of HIV-1 NC71. Two structural forms of tRNA(mixed), which differ in their degree of tertiary structure, were assayed for their susceptibility to denaturation by NC71. Five molar monomer equivalents of NC71 are required to denature the "inactive" tRNA in the absence of Mg2+. A Zn(II)-free, oxidized form of NC71 was also shown to unwind inactive tRNA with the same efficiency and stoichiometry. The detailed spectral changes which occur on NC-induced denaturation closely mimic temperature-induced denaturation of inactive tRNA(mixed). The prototype helix-destabilizing protein, T4 gene 32 protein, is unable to unwind this form of tRNA under the same conditions. The stoichiometry of unwinding of inactive tRNA by NC71 is consistent with the site size determined with poly(A). An "active" form of tRNA(mixed), prepared by thermal denaturation and refolding of the inactive form with Mg2+, proved less susceptible to both temperature and NC71-induced unwinding. The mechanistic implications of these findings on the reported biochemical activities of RNA:RNA annealing and replication primer tRNA positioning by NC are discussed.
所有动物逆转录病毒的核衣壳蛋白（NC）由gag基因编码，是核心核糖核蛋白复合体的主要结构蛋白，在成熟病毒颗粒中与基因组RNA结合。NC也被认为在逆转录过程中发挥一种或多种辅助作用。来自1型人类免疫缺陷病毒（HIV-1）的成熟NC（p7NC）是一种由71个氨基酸组成的碱性蛋白，含有两个Cys3His锌（II）逆转录病毒型锌指结构域。在此，我们描述了从大肠杆菌中可诱导的噬菌体T7 RNA聚合酶启动子亚克隆和表达HIV-1 NC（称为NC71）的过程。通过原子吸收法测定，纯化的NC71可以与2 g.at的锌（II）可逆地重组。紫外圆二色光谱已被用于表征高度纯化的NC71与RNA同聚核苷酸聚（A）和大肠杆菌tRNA（混合）之间的复合物。在聚（A）上，Zn2 NC71的特征是表观位点大小n = 15 +/- 3个核苷酸，具有高亲和力（Kapp = 3 x 10(7) M-1）和适度的协同性（ω约为170 +/- 25）结合。使用大肠杆菌tRNA种类的混合物（混合tRNA）来探测HIV-1 NC71结合后tRNA中诱导的构象变化。测定了两种三级结构程度不同的混合tRNA的结构形式对NC71变性的敏感性。在没有Mg2+的情况下，需要5摩尔单体当量的NC71才能使“无活性”tRNA变性。还显示无锌（II）的氧化形式的NC71以相同的效率和化学计量解开无活性tRNA。NC诱导变性时发生的详细光谱变化与无活性混合tRNA的温度诱导变性非常相似。原型螺旋不稳定蛋白，T4基因32蛋白，在相同条件下无法解开这种形式的tRNA。NC71解开无活性tRNA的化学计量与用聚（A）测定的位点大小一致。通过热变性并在Mg2+存在下重新折叠无活性形式制备的“活性”混合tRNA对温度和NC71诱导的解旋都不太敏感。讨论了这些发现对报道的RNA：RNA退火和NC定位复制引物tRNA的生化活性的机制意义。
