INDEPENDENT IN-VITRO ASSEMBLY OF A RIBONUCLEOPROTEIN PARTICLE CONTAINING THE 3' DOMAIN OF 16S RIBOSOMAL-RNA
INDEPENDENT IN-VITRO ASSEMBLY OF A RIBONUCLEOPROTEIN PARTICLE CONTAINING THE 3' DOMAIN OF 16S RIBOSOMAL-RNA
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DOI：
10.1073/pnas.91.17.7884
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发表时间：
1994-08-16
影响因子：
11.1
通讯作者：
NOLLER, HF
中科院分区：
文献类型：
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作者：
SAMAHA, RR;OBRIEN, B;NOLLER, HF
大肠杆菌核糖体小亚基(30S)由21个蛋白质和一个1542个核苷酸的16S rRNA组成，其二级结构分为三个结构域。16S rRNA的3‘端结构域(残基923-1542)的体外转录本与30S核糖体蛋白有效地组装形成致密的核糖核蛋白(RNP)颗粒。电子显微镜下观察到的孤立颗粒呈球形，大小和形状与30S核糖体亚基的头部相似。二维凝胶分析表明，S3、S7、S9、S10、S13、S14和S19蛋白以及少量的S2存在，免疫电子显微镜和中子衍射表明，所有这些蛋白都定位在30S亚基的头部，属于S7组装家族。有趣的是，被认为只与5‘结构域相互作用的蛋白S4也被重复地发现与大量的颗粒相关。对组装颗粒中RNA的化学探测揭示了特征的切割保护模式，表明蛋白质与3‘-结构域RNA组装的方式类似于它们与16S rRNA组装的方式，尽管后面的一些组装步骤似乎不完整。这些结果表明，16S rRNA的3‘结构域确实可以独立于30S亚基的其余部分组装成与其在核糖体中的结构相似的颗粒。此外，组装的颗粒能够结合壮观霉素，其亲和力与30S亚基的亲和力相当。
Small (30S) subunits of Escherichia coli ribosomes are composed of 21 proteins and a 1542-nucleotide 16S rRNA, whose secondary structure is divided into three domains. An in vitro transcript of the 3' domain of 16S rRNA (residues 923-1542), assembles efficiently with 30S ribosomal proteins to form a compact ribonucleoprotein (RNP) particle. Isolated particles examined under the electron microscope have a globular appearance, similar in size and shape to the head of the 30S ribosomal subunit. Two-dimensional gel analysis of the particles indicates the presence of proteins S3, S7, S9, S10, S13, S14, and S19 and smaller amounts of S2, all of which have been localized to the head of the 30S subunit by immunoelectron microscopy and neutron diffraction and belong to the S7 assembly family. Interestingly, protein S4, which is believed to interact exclusively with the 5' domain, is also reproducibly found associated with the particles in significant amounts. Chemical probing of the RNA in the assembled particle reveals characteristic cleavage protection patterns, showing that the proteins assemble with the 3'-domain RNA similarly to the way in which they assemble with 16S rRNA, although some of the later steps of assembly appear to be incomplete. These results show that the 3' domain of 16S rRNA can indeed assemble independently of the rest of the 30S subunit into a particle that resembles its structure in the ribosome. In addition, the assembled particles are able to bind spectinomycin with an affinity comparable to that of 30S subunits.