Reconstitution of nucleoprotein complexes with mammalian heterogeneous nuclear ribonucleoprotein (hnRNP) core proteins.
Reconstitution of nucleoprotein complexes with mammalian heterogeneous nuclear ribonucleoprotein (hnRNP) core proteins.
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DOI：
10.1083/jcb.97.1.99
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发表时间：
1983-07
影响因子：
7.8
通讯作者：
Martin, T E
中科院分区：
文献类型：
--
作者：
Pullman, J M;Martin, T E
真核细胞中新转录的异质核RNA(HnRNA)被蛋白质结合，产生大的核糖核蛋白(RNP)纤维，其固有亚结构主要由相对均一的约20 nm的30S颗粒组成，其中包含34,000-38,000摩尔重量的核心多肽。为了确定这组蛋白质是否足以组装天然的珠状核蛋白结构，我们通过离子清洗剂脱氧胆酸钠处理从小鼠腹水细胞中分离出30S hnRNP成为其组成蛋白和RNA，然后通过添加Triton X-100来分离脱氧胆酸盐来重组这一复合体。通过蔗糖梯度离心法、监测紫外线吸光度、蛋白质组成和放射性标记的核酸，以及电子显微镜来检测解离和重组。内源RNA被消化，并与等量的外源RNA或单链DNA进行RNP复合体的重组。通过蔗糖梯度和电子显微镜分析，这些复合体完全由n个30S亚基组成，其中n是添加的核酸的长度除以一个天然的30S复合体(约1,000个核苷酸)结合的核酸的长度。当重组混合物中的核酸：蛋白质化学计量比不同时，只形成由30S亚基组成的复合体，多余的蛋白质或核酸保持不结合。这些结果强烈表明，核心蛋白决定了30S亚基的基本结构特性，从而决定了hnRNP的基本结构特性。使用模型核酸分子在体外构建RNP复合体将有助于进一步研究mRNA的加工。
Newly transcribed heterogeneous nuclear RNA (hnRNA) in the eucaryote cell nucleus is bound by proteins, giving rise to large ribonucleoprotein (RNP) fibrils with an inherent substructure consisting largely of relatively homogeneous approximately 20-nm 30S particles, which contain core polypeptides of 34,000-38,000 mol wt. To determine whether this group of proteins was sufficient for the assembly of the native beaded nucleoprotein structure, we dissociated 30S hnRNP purified from mouse ascites cells into their component proteins and RNA by treatment with the ionic detergent sodium deoxycholate and then reconstituted this complex by addition of Triton X-100 to sequester the deoxycholate. Dissociation and reassembly were assayed by sucrose gradient centrifugation, monitoring UV absorbance, protein composition, and radiolabeled nucleic acid, and by electron microscopy. Endogenous RNA was digested and reassembly of RNP complexes carried out with equivalent amounts of exogenous RNA or single-stranded DNA. These complexes are composed exclusively of groups of n 30S subunits, as determined by sucrose gradient and electron microscope analysis, where n is the length of the added nucleic acid divided by the length of nucleic acid bound by one native 30S complex (about 1,000 nucleotides). When the nucleic acid: protein stoichiometry in the reconstitution mixture was varied, only complexes composed of 30S subunits were formed; excess protein or nucleic acid remained unbound. These results strongly suggest that core proteins determine the basic structural properties of 30S subunits and hence of hnRNP. In vitro construction of RNP complexes using model nucleic acid molecules should prove useful to the further study of the processing of mRNA.